DNA diagnostics based on mass spectrometry

ABSTRACT

Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and mutations in the molecules are provided.

RELATED APPLICATIONS

[0001] This application is a continuation of allowed U.S. applicationSer. No. 09/879,341, filed Jun. 11, 2001. This application also is acontinuation of U.S. application Ser. No. 09/796,416, filed Feb. 28,2001, now U.S. Pat. No. 6,500,621, and is a continuation of U.S.application Ser. No. 09/495,444, filed Jan. 31, 2000, now U.S. Pat. No.6,300,076, and of U.S. application Ser. No. 09/504,245, filed Feb. 15,2000, now U.S. Pat. No. 6,221,605. This application also is acontinuation of U.S. application Ser. No. 09/287,679, filed Apr. 6,1999, now U.S. Pat. No. 6,258,538. This application is also acontinuation of U.S. application Ser. No. 08/617,256, filed Mar. 18,1996 and now U.S. Pat. No. 6,043,031. This application is also acontinuation-in-part of U.S. application Ser. No. 08/406,199 filed Mar.17, 1995, and now U.S. Pat. No. 5,605,798. U.S. application Ser. No.08/617,256 is a continuation-in-part of U.S. application Ser. No.08/406,199. The subject matter of each of these applications is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The genetic information of all living organisms (e.g. animals,plants and microorganisms) is encoded in deoxyribonucleic acid (DNA). Inhumans, the complete genome is comprised of about 100,000 genes locatedon 24 chromosomes (The Human Genome, T. Strachan, BIOS ScientificPublishers, 1992). Each gene codes for a specific protein which afterits expression via transcription and translation, fulfills a specificbiochemical function within a living cell. Changes in a DNA sequence areknown as mutations and can result in proteins with altered or in somecases even lost biochemical activities; this in turn can cause geneticdisease. Mutations include nucleotide deletions, insertions oralterations (i.e. point mutations). Point mutations can be either“missense”, resulting in a change in the amino acid sequence of aprotein or “nonsense” coding for a stop codon and thereby leading to atruncated protein.

[0003] More than 3000 genetic diseases are currently known (Human GenomeMutations, D. N. Cooper and M. Krawczak, BIOS Publishers, 1993),including hemophilias, thalassemias, Duchenne Muscular Dystrophy (DMD),Huntington's Disease (HD), Alzheimer's Disease and Cystic Fibrosis (CF).In addition to mutated genes, which result in genetic disease, certainbirth defects are the result of chromosomal abnormalities such asTrisomy 21 (Down's Syndrome), Trisomy 13 (Patau Syndrome), Trisomy 18(Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sexchromosome aneuploidies such as Klienfelter's Syndrome (XXY). Further,there is growing evidence that certain DNA sequences may predispose anindividual to any of a number of diseases such as diabetes,arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g.colorectal, breast, ovarian, lung).

[0004] Viruses, bacteria, fungi and other infectious organisms containdistinct nucleic acid sequences, which are different from the sequencecontained in the host cell. Therefore, infectious organisms can also bedetected and identified based on their specific DNA sequences.

[0005] Since the sequence of about 16 nucleotides is specific onstatistical grounds even for the size of the human genome, relativelyshort nucleic acid sequences can be used to detect normal and defectivegenes in higher organisms and to detect infectious microorganisms (e.g.bacteria, fungi, protists and yeast) and viruses. DNA sequences can evenserve as a fingerprint for detection of different individuals within thesame species. (Thompson, J. S. and M. W. Thompson, eds., Genetics inMedicine, W. B. Saunders Co., Philadelphia, Pa. (1986).

[0006] Several methods for detecting DNA are currently being used. Forexample, nucleic acid sequences can be identified by comparing themobility of an amplified nucleic acid fragment with a known standard bygel electrophoresis, or by hybridization with a probe, which iscomplementary to the sequence to be identified. Identification, however,can only be accomplished if the nucleic acid fragment is labeled with asensitive reporter function (e.g. radioactive (³²P, ³⁵S), fluorescent orchemiluminescent). However, radioactive labels can be hazardous and thesignals they produce decay over time. Non-isotopic labels (e.g.fluorescent) suffer from a lack of sensitivity and fading of the signalwhen high intensity lasers are being used. Additionally, performinglabeling, electrophoresis and subsequent detection are laborious,time-consuming and error-prone procedures. Electrophoresis isparticularly error-prone, since the size or the molecular weight of thenucleic acid cannot be directly correlated to the mobility in the gelmatrix. It is known that sequence specific effects, secondary structuresand interactions with the gel matrix are causing artifacts.

[0007] In general, mass spectrometry provides a means of “weighing”individual molecules by ionizing the molecules in vacuo and making them“fly” by volatilization. Under the influence of combinations of electricand magnetic fields, the ions follow trajectories depending on theirindividual mass (m) and charge (z). In the range of molecules with lowmolecular weight, mass spectrometry has long been part of the routinephysical-organic repertoire for analysis and characterization of organicmolecules by the determination of the mass of the parent molecular ion.In addition, by arranging collisions of this parent molecular ion withother particles (e.g. argon atoms), the molecular ion is fragmentedforming secondary ions by the so-called collision induced dissociation(CID). The fragmentation pattern/pathway very often allows thederivation of detailed structural information. Many applications of massspectrometric methods are known in the art, particularly in biosciences,and can be found summarized in Methods of Enzymology, Vol. 193:“MassSpectrometry” (J. A. McCloskey, editor), 1990, Academic Press, New York.

[0008] Due to the apparent analytical advantages of mass spectrometry inproviding high detection sensitivity, accuracy of mass measurements,detailed structural information by CID in conjunction with an MS/MSconfiguration and speed, as well as on-line data transfer to a computer,there has been considerable interest in the use of mass spectrometry forthe structural analysis of nucleic acids. Recent reviews summarizingthis field include K. H. Schram, “Mass Spectrometry of Nucleic AcidComponents, Biomedical Applications of Mass Spectrometry” 34, 203-287(1990); and P. F. Crain, “Mass Spectrometric Techniques in Nucleic AcidResearch, “Mass Spectrometry Reviews 9, 505-554 (1990).

[0009] However, nucleic acids are very polar biopolymers that are verydifficult to volatilize. Consequently, mass spectrometric detection hasbeen limited to low molecular weight synthetic oligonucleotides bydetermining the mass of the parent molecular ion and through this,confirming the already known oligonucleotide sequence, or alternatively,confirming the known sequence through the generation of secondary ions(fragment ions) via CID in the MS/MS configuration utilizing, inparticular, for the ionization and volatilization, the method of fastatomic bombardment (FAB mass spectrometry) or plasma desorption (PD massspectrometry). As an example, the application of FAB to the analysis ofprotected dimeric blocks for chemical synthesis of oligodeoxynucleotideshas been described (Wolter et al. Biomedical Environmental MassSpectrometry 14: 111-116 (1987)).

[0010] Two more recent ionizations/desorption techniques areelectrospray/ionspray (ES) and matrix-assisted laserdesorption/ionization (MALDI). ES mass spectrometry has been introducedby Yamashita et al. (J. Phys. Chem. 88: 4451-59 (1984); PCT ApplicationNo. WO 90/14148) and current applications are summarized in recentreview articles (R. D. Smith et al., Anal. Chem. 62: 882-89 (1990) andB. Ardrey, Electrospray Mass Spectrometry, Spectroscopy Europe 4: 10-18(1992)). The molecular weights of a tetradecanucleotide (Covey et al.“The Determination of Protein, Oligonucleotide and Peptide MolecularWeights by Ionspray Mass Spectrometry,” Rapid Communications in MassSpectrometry 2: 249-256 (1988)), and of a 21-mer (Methods in Enzymology193, “Mass Spectrometry” (McCloskey, editor), p. 425, 1990, AcademicPress, New York) have been published. As a mass analyzer, a quadrupoleis most frequently used. The determination of molecular weights infemtomole amounts of sample is very accurate due to the presence ofmultiple ion peaks which all could be used for the mass calculation.

[0011] MALDI mass spectrometry, in contrast, can be particularlyattractive when a time-of-flight (TOF) configuration is used as a massanalyzer. The MALDI-TOF mass spectrometry has been introduced byHillenkamp et al. (“Matrix Assisted UV-Laser Desorption/Ionization: ANew Approach to Mass Spectrometry of Large Biomolecules,” BiologicalMass Spectrometry (Burlingame and McCloskey, editors), Elsevier SciencePublishers, Amsterdam, pp. 49-60, 1990). Since, in most cases, nomultiple molecular ion peaks are produced with this technique, the massspectra, in principle, look simpler compared to ES mass spectrometry.

[0012] Although DNA molecules up to a molecular weight of 410,000daltons have been desorbed and volatilized (Nelson et al.,“Volatilization of High Molecular Weight DNA by Pulsed Laser Ablation ofFrozen Aqueous Solutions, Science 246: 1585-87 (1989)), this techniquehas so far only shown very low resolution (oligothymidylic acids up to18 nucleotides, Huth-Fehre et al., Rapid Communications in MassSpectrometry 6: 209-13 (1992); DNA fragments up to 500 nucleotidase inlength, Tang, K. et al., Rapid Communications in Mass Spectrometry 8:727-730 (1994); and a double-stranded DNA of 28 base pairs (Williams etal., “Time-of Flight Mass Spectrometry of Nucleic Acids by LaserAblation and Ionization from a Frozen Aqueous Matrix,” RapidCommunications in Mass Spectrometry 4: 348-351 (1990)).

[0013] Japanese Patent No. 59-131909 describes an instrument, whichdetects nucleic acid fragments separated either by electrophoresis,liquid chromatography or high speed gel filtration. Mass spectrometricdetection is achieved by incorporating into the nucleic acids, atomswhich normally do not occur in DNA such as S, Br, I or Ag, Au, Pt, Os,Hg.

SUMMARY OF THE INVENTION

[0014] The instant invention provides mass spectrometric processes fordetecting a particular nucleic acid sequence in a biological sample.Depending on the sequence to be detected, the processes can be used, forexample, to diagnose (e.g. prenatally or postnatally) a genetic diseaseor chromosomal abnormality; a predisposition to a disease or condition(e.g. obesity, atherosclerosis, cancer), or infection by a pathogenicorganism (e.g. virus, bacteria, parasite or fungus); or to provideinformation relating to identity, heredity, or compatibility (e.g. HLAphenotyping).

[0015] In a first embodiment, a nucleic acid molecule containing thenucleic acid sequence to be detected (i.e. the target) is initiallyimmobilized to a solid support. Immobilization can be accomplished, forexample, based on hybridization between a portion of the target nucleicacid molecule, which is distinct from the target detection site and acapture nucleic acid molecule, which has been previously immobilized toa solid support. Alternatively, immobilization can be accomplished bydirect bonding of the target nucleic acid molecule and the solidsupport. Preferably, there is a spacer (e.g. a nucleic acid molecule)between the target nucleic acid molecule and the support. A detectornucleic acid molecule (e.g. an oligonucleotide or oligonucleotidemimetic), which is complementary to the target detection site can thenbe contacted with the target detection site and formation of a duplex,indicating the presence of the target detection site can be detected bymass spectrometry. In preferred embodiments, the target detection siteis amplified prior to detection and the nucleic acid molecules areconditioned. In a further preferred embodiment, the target detectionsequences are arranged in a format that allows multiple simultaneousdetections (multiplexing), as well as parallel processing usingoligonucleotide arrays (“DNA chips”).

[0016] In a second embodiment, immobilization of the target nucleic acidmolecule is an optional rather than a required step. Instead, once anucleic acid molecule has been obtained from a biological sample, thetarget detection sequence is amplified and directly detected by massspectrometry. In preferred embodiments, the target detection site and/orthe detector oligonucleotides are conditioned prior to massspectrometric detection. In another preferred embodiment, the amplifiedtarget detection sites are arranged in a format that allows multiplesimultaneous detections (multiplexing), as well as parallel processingusing oligonucleotide arrays (“DNA chips”).

[0017] In a third embodiment, nucleic acid molecules which have beenreplicated from a nucleic acid molecule obtained from a biologicalsample can be specifically digested using one or more nucleases (usingdeoxyribonucleases for DNA or ribonucleases for RNA) and the fragmentscaptured on a solid support carrying the corresponding complementarysequences. Hybridization events and the actual molecular weights of thecaptured target sequences provide information on whether and wheremutations in the gene are present. The array can be analyzed spot byspot using mass spectrometry. DNA can be similarly digested using acocktail of nucleases including restriction endonucleases. In apreferred embodiment, the nucleic acid fragments are conditioned priorto mass spectrometric detection.

[0018] In a fourth embodiment, at least one primer with 3′ terminal basecomplementarity to a an allele (mutant or normal) is hybridized with atarget nucleic acid molecule, which contains the allele. An appropriatepolymerase and a complete set of nucleoside triphosphates or only one ofthe nucleoside triphosphates are used in separate reactions to furnish adistinct extension of the primer. Only if the primer is appropriatelyannealed (i.e. no 3′ mismatch) and if the correct (i.e. complementary)nucleotide is added, will the primer be extended. Products can beresolved by molecular weight shifts as determined by mass spectrometry.

[0019] In a fifth embodiment, a nucleic acid molecule containing thenucleic acid sequence to be detected (i.e. the target) is initiallyimmobilized to a solid support. Immobilization can be accomplished, forexample, based on hybridization between a portion of the target nucleicacid molecule, which is distinct from the target detection site and acapture nucleic acid molecule, which has been previously immobilized toa solid support. Alternatively, immobilization can be accomplished bydirect bonding of the target nucleic acid molecule and the solidsupport. Preferably, there is a spacer (e.g. a nucleic acid molecule)between the target nucleic acid molecule and the support. A nucleic acidmolecule that is complementary to a portion of the target detection sitethat is immediately 5′ of the site of a mutation is then hybridized withthe target nucleic acid molecule. The addition of a complete set ofdideoxynucleosides or 3′-deoxynucleoside triphosphates (e.g. pppAdd,pppTdd, pppCdd and pppGdd) and a DNA dependent DNA polymerase allows forthe addition only of the one dideoxynucleoside or 3′-deoxynucleosidetriphosphate that is complementary to X. The hybridization product canthen be detected by mass spectrometry.

[0020] In a sixth embodiment, a target nucleic acid is hybridized with acomplementary oligonucleotides that hybridize to the target within aregion that includes a mutation M. The heteroduplex is than contactedwith an agent that can specifically cleave at an unhybridized portion(e.g. a single strand specific endonuclease), so that a mismatch,indicating the presence of a mutation, results in a the cleavage of thetarget nucleic acid. The two cleavage products can then be detected bymass spectrometry.

[0021] In a seventh embodiment, which is based on the ligase chainreaction (LCR), a target nucleic acid is hybridized with a set ofligation educts and a thermostable DNA ligase, so that the ligase eductsbecome covalently linked to each other, forming a ligation product. Theligation product can then be detected by mass spectrometry and comparedto a known value. If the reaction is performed in a cyclic manner, theligation product obtained can be amplified to better facilitatedetection of small volumes of the target nucleic acid. Selection betweenwildtype and mutated primers at the ligation point can result in adetection of a point mutation.

[0022] The processes of the invention provide for increased accuracy andreliability of nucleic acid detection by mass spectrometry. In addition,the processes allow for rigorous controls to prevent false negative orpositive results. The processes of the invention avoid electrophoreticsteps, labeling and subsequent detection of a label. In fact it isestimated that the entire procedure, including nucleic acid isolation,amplification, and mass spec analysis requires only about 2-3 hourstime. Therefore the instant disclosed processes of the invention arefaster and less expensive to perform than existing DNA detectionsystems. In addition, because the instant disclosed processes allow thenucleic acid fragments to be identified and detected at the same time bytheir specific molecular weights (an unambiguous physical standard), thedisclosed processes are also much more accurate and reliable thancurrently available procedures.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1A is a diagram showing a process for performing massspectrometric analysis on one target detection site (TDS) containedwithin a target nucleic acid molecule (T), which has been obtained froma biological sample. A specific capture sequence (C) is attached to asolid support (SS) via a spacer (S). The capture sequence is chosen tospecifically hybridize with a complementary sequence on the targetnucleic acid molecule (T), known as the target capture site (TCS). Thespacer (S) facilitates unhindered hybridization. A detector nucleic acidsequence (D), which is complementary to the TDS is then contacted withthe TDS. Hybridization between D and the TDS can be detected by massspectrometry.

[0024]FIG. 1B is a diagram showing a process for performing massspectrometric analysis on at least one target detection site (here TDS 1and TDS 2) via direct linkage to a solid support. The target sequence(T) containing the target detection site (TDS 1 and TDS 2) isimmobilized to a solid support via the formation of a reversible orirreversible bond formed between an appropriate functionality (L′) onthe target nucleic acid molecule (T) and an appropriate functionality(L) on the solid support. Detector nucleic acid sequences (here D1 andD2), which are complementary to a target detection site (TDS 1 or TDS 2)are then contacted with the TDS. Hybridization between TDS 1 and D1and/or TDS 2 and D2 can be detected and distinguished based on molecularweight differences.

[0025]FIG. 1C is a diagram showing a process for detecting a wildtype(D^(wt)) and/or a mutant (D^(mut)) sequence in a target (T) nucleic acidmolecule. As in FIG. 1A, a specific capture sequence (C) is attached toa solid support (SS) via a spacer (S). In addition the capture sequenceis chosen to specifically interact with a complementary sequence on thetarget sequence (T), the target capture site (TCS) to be detectedthrough hybridization. However, the target detection site (TDS) includesmutation, X, which changes the molecular weight; mutated targetdetection sites can be distinguished from wildtype by mass spectrometry.Preferably, the detector nucleic acid molecule (D) is designed so thatthe mutation is in the middle of the molecule and therefore would notlead to a stable hybrid if the wildtype detector oligonucleotide(D^(wt)) is contacted with the target detector sequence, e.g. as acontrol. The mutation can also be detected if the mutated detectoroligonucleotide (D^(mut)) with the matching base as the mutated positionis used for hybridization. If a nucleic acid molecule obtained from abiological sample is heterozygous for the particular sequence (i.e.contain both D^(wt) and D^(mut)) both D^(wt) and D^(mut) will be boundto the appropriate strand and the mass difference allows both D^(wt) andD^(mut) to be detected simultaneously.

[0026]FIG. 2 is a diagram showing a process in which several mutationsare simultaneously detected on one target sequence by employingcorresponding detector oligonucleotides. The molecular weightdifferences between the detector oligonucleotides D1, D2 and D3 must belarge enough so that simultaneous detection (multiplexing) is possible.This can be achieved either by the sequence itself (composition orlength) or by the introduction of mass-modifying functionalities M1-M3into the detector oligonucleotide.

[0027]FIG. 3 is a diagram showing still another multiplex detectionformat. In this embodiment, differentiation is accomplished by employingdifferent specific capture sequences which are position-specificallyimmobilized on a flat surface (e.g., a ‘chip array’). If differenttarget sequences T1-Tn are present, their capture sites TCS1-TCSn willinteract with complementary immobilized capture sequences C1-Cn.Detection is achieved by employing appropriately mass differentiateddetector oligonucleotides D1-Dn, which are mass differentiated either bytheir sequences or by mass modifying functionalities M1-Mn.

[0028]FIG. 4 is a diagram showing a format wherein a predesigned targetcapture site (TCS) is incorporated into the target sequence using PCRamplification. Only one strand is captured, the other is removed (e.g.,based on the interaction between biotin and streptavidin coated magneticbeads). If the biotin is attached to primer 1 the other strand can beappropriately marked by a TCS. Detection is as described above throughthe interaction of a specific detector oligonucleotide D with thecorresponding target detection site TDS via mass spectrometry.

[0029]FIG. 5 is a diagram showing how amplification (here ligase chainreaction (LCR)) products can be prepared and detected by massspectrometry. Mass differentiation can be achieved by the mass modifyingfunctionalities (M1 and M2) attached to primers (P1 and P4respectively). Detection by mass spectrometry can be accomplisheddirectly (i.e.) without employing immobilization and target capturingsites (TCS)). Multiple LCR reaction can be performed in parallel byproviding an ordered array of capturing sequences (C). This formatallows separation of the ligation products and spot by spotidentification via mass spectrometry or multiplexing if massdifferentiation is sufficient.

[0030]FIG. 6A is a diagram showing mass spectrometric analysis of anucleic acid molecule, which has been amplified by a transcriptionamplification procedure. An RNA sequence is captured via its TCSsequence, so that wildtype and mutated target detection sites can bedetected as above by employing appropriate detector oligonucleotides(D).

[0031]FIG. 6B is a diagram showing multiplexing to detect two different(mutated) sites on the same RNA in a simultaneous fashion usingmass-modified detector oligonucleotides M1-D1 and M2-D2.

[0032]FIG. 6C is a diagram of a different multiplexing procedure fordetection of specific mutations by employing mass modifieddideoxynucleoside or 3′-deoxynucleoside triphosphates and an RNAdependent DNA polymerase. Alternatively, DNA dependent RNA polymeraseand ribonucleotide triphosphates can be employed. This format allows forsimultaneous detection of all four base possibilities at the site of amutation (X).

[0033]FIG. 7A is a diagram showing a process for performing massspectrometric analysis on one target detection site (TDS) containedwithin a target nucleic acid molecule (T), which has been obtained froma biological sample. A specific capture sequence (C) is attached to asolid support (SS) via a spacer (S). The capture sequence is chosen tospecifically hybridize with a complementary sequence on T known as thetarget capture site (TCS). A nucleic acid molecule that is complementaryto a portion of the TDS is hybridized to the TDS 5′ of the site of amutation (X) within the TDS. The addition of a complete set ofdideoxynucleosides or 3′-deoxynucleoside triphosphates (e.g. pppAdd,pppTdd, pppCdd and pppGdd) and a DNA dependent DNA polymerase allows forthe addition only of the one dideoxynucleoside or 3′-deoxynucleosidetriphosphate that is complementary to X.

[0034]FIG. 7B is a diagram showing a process for performing massspectrometric analysis to determine the presence of a mutation at apotential mutation site (M) within a nucleic acid molecule. This formatallows for simultaneous analysis of both alleles (A) and (B) of a doublestranded target nucleic acid molecule, so that a diagnosis of homozygousnormal, homozygous mutant or heterozygous can be provided. Allele A andB are each hybridized with complementary oligonucleotides ((C) and (D)respectively), that hybridize to A and B within a region that includesM. Each heteroduplex is then contacted with a single strand specificendonuclease, so that a mismatch at M, indicating the presence of amutation, results in the cleavage of (C) and/or (D), which can then bedetected by mass spectrometry.

[0035]FIG. 8 is a diagram showing how both strands of a target DNA canbe prepared for detection using transcription vectors having twodifferent promoters at opposite locations (e.g. the SP 6 and T7promoter). This format is particularly useful for detecting heterozygoustarget detection sites (TDS). Employing the SP 6 or the T7 RNApolymerase both strands could be transcribed separately orsimultaneously. Both RNAs can be specifically captured andsimultaneously detected using appropriately mass-differentiated detectoroligonucleotides. This can be accomplished either directly in solutionor by parallel processing of many target sequences on an ordered arrayof specifically immobilized capturing sequences.

[0036]FIG. 9 is a diagram showing how RNA prepared as described in FIGS.6, 7 and 8 can be specifically digested using one or more ribonucleasesand the fragments captured on a solid support carrying the correspondingcomplementary sequences. Hybridization events and the actual molecularweights of the captured target sequences provide information on whetherand where mutations in the gene are present. The array can be analyzedspot by spot using mass spectrometry. DNA can be similarly digestedusing a cocktail of nucleases including restriction endonucleases.Mutations can be detected by different molecular weights of specific,individual fragments compared to the molecular weights of the wildtypefragments.

[0037]FIG. 10A shows a spectra resulting from the experiment describedin the following Example 1. FIG. 10A-1 shows the absorbance or the26-mer before hybridization. FIG. 10A-2 shows the filtrate of thecentrifugation after hybridization. FIG. 10A-3 shows the results afterthe first wash with 50 mM ammonium citrate. FIG. 10A-4 shows the resultsafter the second wash with 50 mM ammonium citrate.

[0038]FIG. 10B shows a spectra resulting from the experiment describedin the following Example 1 after three washing/centrifugation steps.

[0039]FIG. 10C shows a spectra resulting from the experiment describedin the following Example 1 showing the successful desorption of thehybridized 26-mer off of beads.

[0040]FIG. 11 shows a spectra resulting from the experiment described inthe following Example 1 showing the successful desorption of thehybridized 40-mer. The efficiency of detection suggests that fragmentsmuch longer 40-mers can also be desorbed.

[0041] FIGS. 12A-12C show spectra resulting from the experimentdescribed in the following Example 2 showing the successful desorptionand differentiation of an 18-mer and 19-mer by electrospray massspectrometry: the mixture (12A), peaks resulting from the 18-mer,emphasized (12B) and peaks resulting from the 19-mer, emphasized (12C).

[0042]FIG. 13 is a graphic representation of the process for detectingthe Cystic Fibrosis mutation ΔF508 as described in Example 3; Nindicates normal and M indicats the mutation detection primer orextended primer.

[0043]FIG. 14 is a mass spectrum of the DNA extension product of a ΔF508homozygous normal.

[0044]FIG. 15 is a mass spectrum of the DNA extension product of a ΔF508heterozygous mutant.

[0045]FIG. 16 is a mass spectrum of the DNA extension product of a ΔF508homozygous normal.

[0046]FIG. 17 is a mass spectrum of the DNA extension product of a ΔF508homozygous mutant.

[0047]FIG. 18 is mass spectrum of the DNA extension product of a ΔF508heterozygous mutant.

[0048]FIG. 19 is a graphic representation of various processes forperforming apolipoprotein E genotyping.

[0049] FIGS. 20A-20B show the nucleic acid sequence of normalapolipoprotein E (encoded by the E3 allele) and other isotypes encodedby the E2 and E4 alleles.

[0050]FIG. 21A shows the composite restriction pattern for variousgenotypes of apolipoprotein E.

[0051]FIG. 21B shows the restriction pattern obtained in a 3.5% MetPhorAgarose Gel for various genotypes of apolipoprotein E.

[0052]FIG. 21 C shows the restriction pattern obtained in a 12%polyacrylamide gel for various genotypes of apolipoprotein E.

[0053]FIG. 22A is a chart showing the molecular weights of the 91, 83,72, 48 and 35 base pair fragments obtained by restriction enzymecleavage of the E2, E3, and E4 alleles of apolipoprotein E.

[0054]FIG. 22B is the mass spectra of the restriction product of ahomozygous E4 apolipoprotein E genotype.

[0055]FIG. 23A is the mass spectra of the restriction product of ahomozygous E3 apolipoprotein E genotype.

[0056]FIG. 23B is the mass spectra of the restriction product of a E3/E4apolipoprotein E genotype.

[0057]FIG. 24 is an autoradiograph of a 7.5% polyacrylamide gel in which10% (5 μl) of each PCR was loaded. Sample M: pBR322 Alul digested;sample 1: HBV positive in serological analysis; sample 2: also HBVpositive; sample 3: without serological analysis but with an increasedlevel of transaminases, indicating liver disease; sample 4: HBVnegative; sample 5: HBV positive by serological analysis; sample 6: HBVnegative (−) negative control; (+) positive control). Staining was donewith ethidium bromide.

[0058]FIG. 25A is a mass spectrum of sample 1, which is HBV positive.The signal at 20754 Da represent HBV related PCR product (67nucleotides, calculated mass: 20735 Da). The mass signal at 10390 Darepresents [M+2H]²⁺ signal (calculated: 10378 Da).

[0059]FIG. 25B is a mass spectrum of sample 3, which is HBV negativecorresponding to PCR, serological and dot blot based assays. The PCRproduct is generated only in trace amounts. Nevertheless, it isunambiguously detected at 20751 Da (calculated: 20735 Da). The masssignal at 10397 Da represents the [M+2H]²⁺ molecule ion (calculated:10376 Da).

[0060]FIG. 25C is a mass spectrum of sample 4, which is HBV negative,but CMV positive. As expected, no HBV specific signals could beobtained.

[0061]FIG. 26 shows a part of the E.coli lacl gene with binding sites ofthe complementary oligonucleotides used in the ligase chain reaction(LCR). Here the wildtype sequence is displayed. The mutant contains apoint mutation at bp 191 which is also the site of ligation (bold). Themutation is a C to T transition (G to A, respectively). This leads to aT-G mismatch with oligo A (and A-C mismatch with oligo B. respectively).

[0062]FIG. 27 is a 7.5% polyacrylamide gel stained with ethidiumbromide. M: chain length standard (pUC19 DNA, MspI digested). Lane 1:LCR with wildtype template. Lane 2: LCR with mutant template. Lane 3:(control) LCR without template. The ligation product (50 bp) was onlygenerated in the positive reactive containing wildtype template.

[0063]FIG. 28 is an HPLC chromatogram of two pooled positive LCRs.

[0064]FIG. 29 shows an HPLC chromatogram under the same conditions butusing the mutant template. The small signal of the ligation product isdue to either template-free ligation of the educts or to a ligation at a(G-T, A-C) mismatch. The ‘false positive’ signal is significantly lowerthan the signal of ligation product with wildtype template depicted inFIG. 28. The analysis of ligation educts leads to ‘double-peaks’ becausetwo of the oligonucleotides are 5′-phosphorylated.

[0065]FIG. 30A shows the complex signal pattern obtained by MALDI-TOF-MSanalysis of Pfu DNA-ligase solution. FIG. 30B shows a MALDI-TOF-spectrumof an unpurified LCR. The mass signal 67569 Da probably represents thePfu DNA ligase.

[0066]FIG. 31A shows a MALDI-TOF spectrum of two pooled positive LCRs.The signal at 7523 Da represents unligated oligo A (calculated: 7521 Da)whereas the signal at 15449 Da represents the ligation product(calculated: 15450 Da). The signal at 3774 Da is the [M+2H]²⁺ signal ofoligo A. The signals in the mass range lower than 2000 Da are due to thematrix ions. The spectrum corresponds to lane 1 in FIG. 27 and to thechromatogram in FIG. 28. FIG. 31B shows a spectrum of two poolednegative LCRs (mutant template). The signal at 7517 Da represents oligoA (calculated: 7521 Da).

[0067]FIG. 32 shows a spectrum obtained from two pooled LCRs in whichonly salmon sperm DNA was used as a negative control: only oligo A couldbe detected, as expected.

[0068]FIG. 33A shows a spectrum of two pooled positive LCRs. Thepurification was done with a combination of ultrafiltration andstreptavidin DynaBeads as described in the text. The signal at 15448 Darepresents the ligation product (calculated: 15450 Da). The signal at7527 represents oligo A (calculated: 7521 Da). The signals at 3761 Da isthe [M+2H]²⁺ signal of oligo A, where as the signal at 5140 Da is the[M+3H]²⁺ signal of the ligation product. FIG. 33B shows a spectrum oftwo pooled negative LCRs (without template). The signal at 7514 Darepresents oligo A (calculated: 7521 Da).

[0069]FIG. 34A is a schematic representation of the oligo base extensionof the mutation detection primer b using ddTTP. FIG. 34B is a schematicrepresentation of the oligo base extension of the mutation detectionprimer b using ddCTP. The theoretical mass calculation is given inparenthesis. The sequence shown is part of the exon 10 of the CFTR genethat bears the most common cystic fibrosis mutation ΔF508 and more raremutations Δ507 as well at Ile506Ser.

[0070]FIG. 35A is a MALDI-TOF-MS spectra recorded directly fromprecipitated oligo base extended primers for mutation detection usingddTTP. FIG. 35B is a MALDI-TOF-MS spectra recorded directly fromprecipitated oligo base extended primers for mutation detection usingddCTP. The spectra on the top of each panel (ddTTP or ddCTP,respectively) shows the annealed primer (CF508) without furtherextension reaction. The template of diagnosis is pointed out below eachspectra and the observed/expected molecular mass are written inparenthesis.

[0071]FIG. 36 shows the portion of the sequence of pRFc1 DNA, which wasused as template for PCR amplification of unmodified and 7-deazapurinecontaining 99-mer and 200-mer nucleic acids as well as the sequences ofthe 19-mer primers and the two 18-mer reverse primers.

[0072]FIG. 37 shows the portion of the nucleotide sequence of M13mp18RFI DNA, which was used for PCR amplification of unmodified and7-deazapurine containing 103-mer nucleic acids. Also shown arenucleotide sequences of the 17-mer primers used in the PCR.

[0073]FIG. 38 shows the result of a polyacrylamide gel electrophoresisof PCR products purified and concentrated for MALDI-TOF MS analysis. M:chain length marker, lane 1: 7-deazapurine containing 99-mer PCRproduct, lane 2: unmodified 99-mer, lane 3: 7-deazapurine containing103-mer and lane 4: unmodified 103-mer PCR product.

[0074]FIG. 39: an autoradiogram of polyacrylamide gel electrophoresis ofPCR reactions carried out with 5′-[³²P]-labeled primers 1 and 4. Lanes 1and 2: unmodified and 7-deazapurine modified 200-mer (71123 and 39582counts), lanes 3 and 4: unmodified and 7-deazapurine modified 200-mer(71123 and 39582 counts) and lanes 5 and 6: unmodified and 7-deazapurinemodified 99-mer (173216 and 94400 counts).

[0075]FIG. 40A shows a MALDI-TOF mass spectrum of the unmodified 103-merPCR products (sum of twelve single shot spectra). The mean value of themasses calculated for the two single strands (31768 u and 31759 u) is31763 u. Mass resolution: 18. FIG. 40B shows a MALDI-TOF mass spectrumof 7-deazapurine containing 103-mer PCR product (sum of three singleshot spectra). The mean value of the masses calculated for the twosingle strands (31727 u and 31719 u) is 31723 u. Mass resolution: 67.

[0076] FIGS. 41A1 and 41A2 show a MALDI-TOF mass spectrum of theunmodified 99-mer PCR product (sum of twenty single shot spectra).Values of the masses calculated for the two single strands: 30261 u and30794 u. FIGS. 41B1 and 41B2 show a MALDI-TOF mass spectrum of the7-deazapurine containing 99-mer PCR product (sum of twelve single shotspectra). Values of the masses calculated for the two single strands:30224 u and 30750 u.

[0077]FIG. 42A shows a MALDI-TOF mass spectrum of the unmodified 200-merPCR product (sum of 30 single shot spectra). The mean value of themasses calculated for the two single strands (61873 u and 61595 u) is61734 u. Mass resolution: 28. FIG. 42B shows a MALDI-TOF mass spectrumof 7-deazapurine containing 200-mer PCR product (sum of 30 single shotspectra). The mean value of the masses calculated for the two singlestrands (61772 u and 61514 u) is 61643 u. Mass resolution: 39.

[0078]FIG. 43A shows a MALDI-TOF mass spectrum of 7-deazapurinecontaining 100-mer PCR product with ribomodified primers. The mean valueof the masses calculated for the two single strands (30529 u and 31095u) is 30812 u. FIG. 43B shows a MALDI-TOF mass spectrum of thePCR-product after hydrolytic primer-cleavage. The mean value of themasses calculated for the two single strands (25104 u and 25229 u) is25167 u. The mean value of the cleaved primers (5437 u and 5918 u) is5677 u.

[0079] FIGS. 44A-44D show the MALDI-TOF mass spectrum of the foursequencing ladders obtained from a 39-mer template (SEQ. ID. No. 13),which was immobilized to streptavidin beads via a 3′ biotinylation. A14-mer primer (SEQ. ID. No. 14) was used in the sequencing.

[0080]FIG. 45 shows a MALDI-TOF mass spectrum of a solid statesequencing of a 78-mer template (SEQ. ID. No. 15), which was immobilizedto streptavidin beads via a 3′ biotinylation. A 18-mer primer (SEQ. ID.No. 16) and ddGTP were used in the sequencing.

[0081]FIG. 46 shows a scheme in which duplex DNA probes withsingle-stranded overhang capture specific DNA templates and also serveas primers for solid state sequencing.

[0082]FIG. 47A-47D shows MALDI-TOF mass spectra obtained from a 5′fluorescent labeled 23-mer (SEQ. ID. No. 19) annealed to an 3′biotinylated 18-mer (SEQ. ID. No. 20), leaving a 5-base overhang, whichcaptured a 15-mer template (SEQ. ID. No. 21).

[0083] FIGS. 48A-48D show a stacking fluorogram of the same productsobtained from the reaction described in FIG. 35, but run on aconventional DNA sequencer.

DETAILED DESCRIPTION OF THE INVENTION

[0084] In general, the instant invention provides mass spectrometricprocesses for detecting a particular nucleic acid sequence in abiological sample. As used herein, the term “biological sample” refersto any material obtained from any living source (e.g. human, animal,plant, bacteria, fungi, protist, virus). For use in the invention, thebiological sample should contain a nucleic acid molecule. Examples ofappropriate biological samples for use in the instant invention include:solid materials (e.g. tissue, cell pellets, biopsies) and biologicalfluids (e.g. urine, blood, saliva, amniotic fluid, mouth wash).

[0085] Nucleic acid molecules can be isolated from a particularbiological sample using any of a number of procedures, which arewell-known in the art, the particular isolation procedure chosen beingappropriate for the particular biological sample. For example,freeze-thaw and alkaline lysis procedures can be useful for obtainingnucleic acid molecules from solid materials; heat and alkaline lysisprocedures can be useful for obtaining nucleic acid molecules fromurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Rolff, A et al. PCR: Clinical Diagnostics and Research,Springer (1994).

[0086] To obtain an appropriate quantity of nucleic acid molecules onwhich to perform mass spectrometry, amplification may be necessary.Examples of appropriate amplification procedures for use in theinvention include: cloning (Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989),polymerase chain reaction (PCR) (C. R. Newton and A. Graham, PCR, BIOSPublishers, 1994); ligase chain reaction (LCR) (Wiedmann, M., et al.,PCR Methods Appl. Vol. 3, pp. 57-64 (1994); F. Barnay, Proc. Natl. Acad.Sci USA 88: 189-93 (1991)); strand displacement amplification (SDA) (G.Terrance Walker et al., Nucleic Acids Res. 22: 2670-77 (1994)); andvariations such as RT-PCR (Higuchi, et al., Bio/Technology 11: 1026-1030(1993)), allele-specific amplification (ASA) and transcription basedprocesses.

[0087] To facilitate mass spectrometric analysis, a nucleic acidmolecule containing a nucleic acid sequence to be detected can beimmobilized to a solid support. Examples of appropriate solid supportsinclude beads (e.g. silica gel, controlled pore glass, magnetic,Sephadex/Sepharose, cellulose), flat surfaces or chips (e.g. glass fiberfilters, glass surfaces, metal surface (steel, gold, silver, aluminum,copper and silicon), capillaries, plastic (e.g. polyethylene,polypropylene, polyamide, polyvinylidenedifluoride membranes ormicrotiter plates)); or pins or combs made from similar materialscomprising beads or flat surfaces or beads placed into pits in flatsurfaces such as wafers (e.g. silicon wafers).

[0088] Immobilization can be accomplished, for example, based onhybridization between a capture nucleic acid sequence, which has alreadybeen immobilized to the support and a complementary nucleic acidsequence, which is also contained within the nucleic acid moleculecontaining the nucleic acid sequence to be detected (FIG. 1A). So thathybridization between the complementary nucleic acid molecules is nothindered by the support, the capture nucleic acid can include a spacerregion of at least about five nucleotides in length between the solidsupport and the capture nucleic acid sequence. The duplex formed will becleaved under the influence of the laser pulse and desorption can beinitiated. The solid support-bound base sequence can be presentedthrough natural oligoribo- or oligodeoxyribonucleotide as well asanalogs (e.g. thio-modified phosphodiester or phosphotriester backbone)or employing oligonucleotide mimetics such as PNA analogs (see e.g.Nielsen et al., Science, 254, 1497 (1991)) which render the basesequence less susceptible to enzymatic degradation and hence increasesoverall stability of the solid support-bound capture base sequence.

[0089] Alternatively, a target detection site can be directly linked toa solid support via a reversible or irreversible bond between anappropriate functionality (L′) on the target nucleic acid molecule (T)and an appropriate functionality (L) on the capture molecule (FIG. 1B).A reversible linkage can be such that it is cleaved under the conditionsof mass spectrometry (i.e., a photocleavable bond such as a chargetransfer complex or a labile bond being formed between relatively stableorganic radicals). Furthermore, the linkage can be formed with L′ beinga quaternary ammonium group, in which case, preferably, the surface ofthe solid support carries negative charges which repel the negativelycharged nucleic acid backbone and thus facilitate the desorptionrequired for analysis by a mass spectrometer. Desorption can occureither by the heat created by the laser pulse and/or, depending on L′,by specific absorption of laser energy which is in resonance with the L′chromophore.

[0090] By way of example, the L-L′ chemistry can be of a type ofdisulfide bond (chemically cleavable, for example, by mercaptoethanol ordithioerythritol), a biotin/streptavidin system, a heterobifunctionalderivative of a trityl ether group (Gildea et al., “A VersatileAcid-Labile Linker for Modification of Synthetic Biomolecules,”Tetrahedron Letters 31: 7095 (1990)) which can be cleaved under mildlyacidic conditions as well as under conditions of mass spectrometry, alevulinyl group cleavable under almost neutral conditions with ahydrazinium/acetate buffer, an arginine-arginine or lysine-lysine bondcleavable by an endopeptidase enzyme like trypsin or a pyrophosphatebond cleavable by a pyrophosphatase, or a ribonucleotide bond in betweenthe oligodeoxynucleotide sequence, which can be cleaved, for example, bya ribonuclease or alkali.

[0091] The functionalities, L and L′, can also form a charge transfercomplex and thereby form the temporary L-L′ linkage. Since in many casesthe “charge-transfer band” can be determined by UV/vis spectrometry (seee.g. Organic Charge Transfer Complex by R. Foster, Academic Press,1969), the laser energy can be tuned to the corresponding energy of thecharge-transfer wavelength and, thus, a specific desorption off thesolid support can be initiated. Those skilled in the art will recognizethat several combinations can serve this purpose and that the donorfunctionality can be either on the solid support or coupled to thenucleic acid molecule to be detected or vice versa.

[0092] In yet another approach, a reversible L-L′ linkage can begenerated by homolytically forming relatively stable radicals. Under theinfluence of the laser pulse, desorption (as discussed above) as well asionization will take place at the radical position. Those skilled in theart will recognize that other organic radicals can be selected and that,in relation to the dissociation energies needed to homolytically cleavethe bond between them, a corresponding laser wavelength can be selected(see e.g., Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984).

[0093] An anchoring function L′ can also be incorporated into a targetcapturing sequence (TCS) by using appropriate primers during anamplification procedure, such as PCR (FIG. 4), LCR (FIG. 5) ortranscription amplification (FIG. 6A).

[0094] Prior to mass spectrometric analysis, it may be useful to“condition” nucleic acid molecules, for example to decrease the laserenergy required for volatization and/or to minimize fragmentation.Conditioning is preferably performed while a target detection site isimmobilized. An example of conditioning is modification of thephosphodiester backbone of the nucleic acid molecule (e.g. cationexchange), which can be useful for eliminating peak broadening due to aheterogeneity in the cations bound per nucleotide unit. Contacting anucleic acid molecule with an alkylating agent such as alkyliodide,iodoacetamide, β-iodoethanol, 2,3-epoxy-1-propanol, the monothiophosphodiester bonds of a nucleic acid molecule can be transformed intoa phosphotriester bond. Likewise, phosphodiester bonds may betransformed to uncharged derivatives employing trialkylsilyl chlorides.Further conditioning involves incorporating nucleotides which reducesensitivity for depurination (fragmentation during MS) such as N7- orN9-deazapurine nucleotides, or RNA building blocks or usingoligonucleotide triesters or incorporating phosphorothioate functionswhich are alkylated or employing oligonucleotide mimetics such as PNA.

[0095] For certain applications, it may be useful to simultaneouslydetect more than one (mutated) loci on a particular captured nucleicacid fragment (on one spot of an array) or it may be useful to performparallel processing by using oligonucleotide or oligonucleotide mimeticarrays on various solid supports. “Multiplexing” can be achieved byseveral different methodologies. For example, several mutations can besimultaneously detected on one target sequence by employingcorresponding detector (probe) molecules (e.g. oligonucleotides oroligonucleotide mimetics). However, the molecular weight differencesbetween the detector oligonucleotides D1, D2 and D3 must be large enoughso that simultaneous detection (multiplexing) is possible. This can beachieved either by the sequence itself (composition or length) or by theintroduction of mass-modifying functionalities M1-M3 into the detectoroligonucleotide. (FIG. 2)?

[0096] Mass modifying moieties can be attached, for instance, to eitherthe 5′-end of the oligonucleotide (M¹), to the nucleobase (or bases)(M², M⁷), to the phosphate backbone (M³), and to the 2′-position of thenucleoside (nucleosides) (M⁴, M⁶) or/and to the terminal 3′-position(M⁵). Examples of mass modifying moieties include, for example, ahalogen, an azido, or of the type, XR, wherein X is a linking group andR is a mass-modifying functionality. The mass-modifying functionalitycan thus be used to introduce defined mass increments into theoligonucleotide molecule.

[0097] Here the mass-modifying moiety, M, can be attached either to thenucleobase, M² (in case of the c⁷-deazanucleosides also to C-7, M⁷), tothe triphosphate group at the alpha phosphate, M³, or to the 2′-positionof the sugar ring of the nucleoside triphosphate, M⁴ and M⁶.Furthermore, the mass-modifying functionality can be added so as toaffect chain termination, such as by attaching it to the 3′-position ofthe sugar ring in the nucleoside triphosphate, M⁵. For those skilled inthe art, it is clear that many combinations can serve the purpose of theinvention equally well. In the same way, those skilled in the art willrecognize that chain-elongating nucleoside triphosphates can also bemass-modified in a similar fashion with numerous variations andcombinations in functionality and attachment positions.

[0098] Without limiting the scope of the invention, themass-modification, M, can be introduced for X in XR as well as usingoligo-/-polyethylene glycol derivatives for R. The mass-modifyingincrement in this case is 44, i.e. five different mass-modified speciescan be generated by just changing m from 0 to 4 thus adding mass unitsof 45 (m=0), 89 (m=1), 133 (m=2), 177 (m=3) and 221 (m=4) to the nucleicacid molecule (e.g. detector oligonucleotide (D) or the nucleosidetriphosphates (FIGS. 3 and 6(C), respectively). The oligo/polyethyleneglycols can also be monoalkylated by a lower alkyl such as methyl,ethyl, propyl, isopropyl, t-butyl and the like. A selection of linkingfunctionalities, X, are also illustrated. Other chemistries can be usedin the mass-modified compounds, as for example, those described recentlyin Oligonucleotides and Analogues, A Practical Approach (F. Eckstein,editor, IRL Press, Oxford, 1991).

[0099] In yet another embodiment, various mass-modifyingfunctionalities, R, other than oligo/polyethylene glycols, can beselected and attached via appropriate linking chemistries, X. A simplemass-modification can be achieved by substituting H for halogens like F,Cl, Br and/or I, or pseudohalogens such as SCN, NCS, or by usingdifferent alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl,isopropyl, t-butyl, hexyl, phenyl, substituted phenyl, benzyl, orfunctional groups such as CH₂F, CHF₂, CF₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅),Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃. Yet another mass-modification can be obtainedby attaching homo- or heteropeptides through the nucleic acid molecule(e.g. detector (D)) or nucleoside triphosphates. One example useful ingenerating mass-modified species with a mass increment of 57 is theattachment of oligoglycines, e.g. mass-modifications of 74 (r=1, m=0),131 (r=1, m=2), 188 (r=1, m=3), 245 (r=1, m=4) are achieved. Simpleoligoamides also can be used, e.g., mass-modifications of 74 (r=1, m=0),88 (r=2, m=0), 102 (r=3, m=0), 116 (r=4, m=0), et obtainable. For thoseskilled in the art, it will be obvious that there are numerouspossibilities in addition to those mentioned above.

[0100] As used herein, the superscript O-i designates i+1 massdifferentiated nucleotides, primers or tags. In some instances, thesuperscript O can designate an unmodified species of a particularreactant, and the superscript i can designate the i-th mass-modifiedspecies of that reactant. If, for example, more than one species ofnucleic acids are to be concurrently detected, then i+1 differentmass-modified detector oligonucleotides (D⁰, D¹, . . . D^(i)) can beused to distinguish each species of mass modified detectoroligonucleotides (D) from the others by mass spectrometry.

[0101] Different mass-modified detector oligonucleotides can be used tosimultaneously detect all possible variants/mutants simultaneously (FIG.6B). Alternatively, all four base permutations at the site of a mutationcan be detected by designing and positioning a detector oligonucleotide,so that it serves as a primer for a DNA/RNA polymerase (FIG. 6C). Forexample, mass modifications also can be incorporated during theamplification process.

[0102]FIG. 3 shows a different multiplex detection format, in whichdifferentiation is accomplished by employing different specific capturesequences which are position-specifically immobilized on a flat surface(e.g. a ‘chip array’). If different target sequences T1-Tn are present,their target capture sites TCS1-TCSn will specifically interact withcomplementary immobilized capture sequences C1-Cn. Detection is achievedby employing appropriately mass differentiated detector oligonucleotidesD1-Dn, which are mass differentiated either by their sequences or bymass modifying functionalities M1-Mn.

[0103] Preferred mass spectrometer formats for use in the invention arematrix assisted laser desorption ionization (MALDI), electrospray (ES),ion cyclotron resonance (ICR) and Fourier Transform. For ES, thesamples, dissolved in water or in a volatile buffer, are injected eithercontinuously or discontinuously into an atmospheric pressure ionizationinterface (API) and then mass analyzed by a quadrupole. The generationof multiple ion peaks which can be obtained using ES mass spectrometrycan increase the accuracy of the mass determination. Even more detailedinformation on the specific structure can be obtained using an MS/MSquadrupole configuration.

[0104] In MALDI mass spectrometry, various mass analyzers can be used,e.g., magnetic sector/magnetic deflection instruments in single ortriple quadrupole mode (MS/MS), Fourier transform and time-of-flight(TOF) configurations as is known in the art of mass spectrometry. Forthe desorption/ionization process, numerous matrix/laser combinationscan be used. Ion-trap and reflectron configurations can also beemployed.

[0105] The mass spectrometric processes described above can be used, forexample, to diagnose any of the more than 3000 genetic diseasescurrently known (e.g. hemophilias, thalassemias, Duchenne MuscularDystrophy (DMD), Huntington's Disease (HD), Alzheimer's Disease andCystic Fibrosis (CF)) or to be identified.

[0106] The following Example 3 provides a mass spectrometer method fordetecting a mutation (ΔF508) of the cystic fibrosis transmembraneconductance regulator gene (CFTR), which differs by only three basepairs (900 daltons) from the wild type of CFTR gene. As describedfurther in Example 3, the detection is based on a single-tube,competitive oligonucleotide single base extension (COSBE) reaction usinga pair of primers with the 3′-terminal base complementary to either thenormal or mutant allele. Upon hybridization and addition of a polymeraseand the nucleoside triphosphate one base downstream, only those primersproperly annealed (i.e., no 3′-terminal mismatch) are extended; productsare resolved by molecular weight shifts as determined by matrix assistedlaser desorption ionization time-of-flight mass spectrometry. For thecystic fibrosis ΔF508 polymorphism, 28-mer ‘normal’ (N) and 30-mer‘mutant’ (M) primers generate 29- and 31-mers for N and M homozygotes,respectively, and both for heterozygotes. Since primer and productmolecular weights are relatively low (<10 kDa) and the mass differencebetween these are at least that of a single ˜300 Da nucleotide unit, lowresolution instrumentation is suitable for such measurements.

[0107] In addition to mutated genes, which result in genetic disease,certain birth defects are the result of chromosomal abnormalities suchas Trisomy 21 (Down's syndrome), Trisomy 13 (Platau Syndrome), Trisomy18 (Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sexchromosome aneuploidies such as Klienfelter's Syndrome (XXY).

[0108] Further, there is growing evidence that certain DNA sequences maypredispose an individual to any of a number of diseases such asdiabetes, arteriosclerosis, obesity, various autoimmune diseases andcancer (e.g. colorectal, breast, ovarian, lung); chromosomal abnormality(either prenatally or postnatally); or a predisposition to a disease orcondition (e.g. obesity, atherosclerosis, cancer). Also, the detectionof “DNA fingerprints”, e.g. polymorphisms, such as “microsatellitesequences”, are useful for determining identity or heredity (e.g.paternity or maternity).

[0109] The following Example 4 provides a mass spectrometer method foridentifying any of the three different isoforms of human apolipoproteinE, which are coded by the E2, E3 and E4 alleles. Here the molecularweights of DNA fragments obtained after restriction with appropriaterestriction endonucleases can be used to detect the presence of amutation.

[0110] Depending on the biological sample, the diagnosis for a geneticdisease, chromosomal aneuploidy or genetic predisposition can bepreformed either pre- or post-natally.

[0111] Viruses, bacteria, fungi and other organisms contain distinctnucleic acid sequences, which are different from the sequences containedin the host cell. Detecting or quantitating nucleic acid sequences thatare specific to the infectious organism is important for diagnosing ormonitoring infection. Examples of disease causing viruses that infecthumans and animals and which may be detected by the disclosed processesinclude: Retroviridae (e.g., human immunodeficiency viruses, such asHIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, see Ratner, L.et al., Nature 313: 227-284 (1985); and Wain Hobson, S., et al, Cell 40:9-17 (1985)); HIV-2 (see Guyader et al., Nature 328: 662-669 (1987);European Patent Publication No. 0 269 520; Chakraborti et al., Nature328: 543-547 (1987); and European Patent Application No. 0 655 501); andother isolates, such as HIV-LP (International Publication No. WO94/00562 entitled “A Novel Human Immunodeficiency Virus”; Picornaviridae(e.g., polio viruses, hepatitis A virus, (Gust, I. D., et al.,Intervirology, Vol. 20, pp. 1-7 (1983); entero viruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains thatcause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses,rubella viruses); Flaviridae (e.g., dengue viruses, encephalitisviruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviursesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses′); Poxviridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (e.g., African swine fever virus); andunclassified viruses (e.g., the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

[0112] Examples of infectious bacteria include: Helicobacter pyloris,Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

[0113] Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.

[0114] The following Example 5 provides a nested PCR and massspectrometer based method that was used to detect hepatitis B virus(HBV) DNA in blood samples. Similarly, other blood-borne viruses (e.g.,HIV-1, HIV-2, hepatitis C virus (HCV), hepatitis A virus (HAV) and otherhepatitis viruses (e.g., non-A-non-B hepatitis, hepatitis G, hepatitisE), cytomegalovirus, and herpes simplex virus (HSV)) can be detectedeach alone or in combination based on the methods described herein.

[0115] Since the sequence of about 16 nucleotides is specific onstatistical grounds (even for a genome as large as the human genome),relatively short nucleic acid sequences can be used to detect normal anddefective genes in higher organisms and to detect infectiousmicroorganisms (e.g. bacteria, fungi, protists and yeast) and viruses.DNA sequences can even serve as a fingerprint for detection of differentindividuals within the same species. (Thompson, J. S. and M. W.Thompson, eds., Genetics in Medicine, W. B. Saunders Co., Philadelphia,Pa. (1986).

[0116] One process for detecting a wildtype (D^(wt)) and/or a mutant(D^(mut)) sequence in a target (T) nucleic acid molecule is shown inFIG. 1C. A specific capture sequence (C) is attached to a solid support(ss) via a spacer (S). In addition, the capture sequence is chosen tospecifically interact with a complementary sequence on the targetsequence (T), the target capture site (TCS) to be detected throughhybridization. However, if the target detection site (TDS) includes amutation, X, which increases or decreases the molecular weight, mutatedTDS can be distinguished from wildtype by mass spectrometry. Forexample, in the case of an adenine base (dA) insertion, the differencein molecular weights between D^(wt) and D^(mut) would be about 314daltons.

[0117] Preferably, the detector nucleic acid (D) is designed such thatthe mutation would be in the middle of the molecule and the flankingregions are short enough so that a stable hybrid would not be formed ifthe wildtype detector oligonucleotide (D^(wt)) is contacted with themutated target detector sequence as a control. The mutation can also bedetected if the mutated detector oligonucleotide (D^(mut)) with thematching base at the mutated position is used for hybridization. If anucleic acid obtained from a biological sample is heterozygous for theparticular sequence (i.e. contain both D^(wt) and D^(mut)), both D^(wt)and D^(mut) will be bound to the appropriate strand and the massdifference allows by D^(wt) and D^(mut) to be detected simultaneously.

[0118] The process of this invention makes use of the known sequenceinformation of the target sequence and known mutation sites, althoughnew mutations can also be detected. For example, as shown in FIG. 8,transcription of a nucleic acid molecule obtained from a biologicalsample can be specifically digested using one or more nucleases and thefragments captured on a solid support carrying the correspondingcomplementary nucleic acid sequences. Detection of hybridization and themolecular weights of the captured target sequences provide informationon whether and where in a gene a mutation is present. Alternatively, DNAcan be cleaved by one or more specific endonucleases to form a mixtureof fragments. Comparison of the molecular weights between wildtype andmutant fragment mixtures results in mutation detection.

[0119] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications (including internationalpatent application Publication Number WO 94/16101 and U.S. Pat. No.5,605,798, entitled DNA Sequencing by Mass Spectrometry by H. Köster;and international patent application Publication Number WO 94/21822 andU.S. Pat. No. 5,622,824, entitled “DNA Sequencing by Mass SpectrometryVia Exonuclease Degradation” by H. Köster), and co-pending patentapplications, (including U.S. patent application Ser. No. 08/406,199,now U.S. Pat. No. 5,605,798 entitled DNA Diagnostics Based on MassSpectrometry by H. Köster), as cited throughout this application arehereby expressly incorporated by reference.

EXAMPLE 1 MALDI-TOF Desorption of Oligonucleotides Directly on SolidSupports

[0120] 1 g CPC (Controlled Pore Glass) was functionalized with3-(triethoxysilyl)-epoxypropan to form OH-groups on the polymer surface.A standard oligonucleotide synthesis with 13 mg of the OH-CPG on a DNAsynthesizer (Milligen, Model 7500) employingβ-cyanoethyl-phosphoamidites (Sinha et al., Nucleic Acids Res. 12: 4539(1984)) and TAC N-protecting groups (Köster et al., Tetrahedron 37: 362(1981)) was performed to synthesize a 3′-T₅-50-mer oligonucleotidesequence in which 50 nucleotides are complementary to a “hypothetical”50-mer sequence. T₅ serves as a spacer. Deprotection with saturatedammonia in methanol at room temperature for 2 hours furnished accordingto the determination of the DMT group CPG which contained about 10 μmol55-mer/g CPG. This 55-mer served as a template for hybridizations with a26-mer (with 5′-DMT group) and a 40-mer (without DMT group). Thereaction volume is 100 μl and contains about 1 nmol CPG bound 55-mer astemplate, an equimolar amount of oligonucleotide in solution (26-mer or40-mer) in 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂ and 25 mM NaCl. Themixture was heated for 10′ at 65° C. and cooled at 37° C. during 30′(annealing). The oligonucleotide which has not been hybridized to thepolymer-bound template were removed by centrifugation and threesubsequent washing/centrifugation steps with 100 μl each of ice-cold 50mM ammonium citrate. The beads were air-dried and mixed with matrixsolution (3-hydroxypicolinic acid/10 mM ammonium citrate inacetonitrile/water, 1:1), and analyzed by MALDI-TOF mass spectrometry.The results are presented in FIGS. 10A-10C and 11.

EXAMPLE 2 Electrospray (ES) Desorption and Differentiation of an 18-merand 19-mer

[0121] DNA fragments at a concentration of 50 pmole/μl in 2-propanol/10mM ammonium carbonate (1/9, v/v) were analyzed simultaneously by anelectrospray mass spectrometer.

[0122] The successful desorption and differentiation of an 18-mer and19-mer by electrospray mass spectrometry is shown in FIGS. 12A-12C.

EXAMPLE 3 Detection of the Cystic Fibrosis Mutation, ΔF508, by SingleStep Dideoxy Extension and Analysis by MALDI-TOF Mass SpectrometryMaterial and Methods

[0123] PCR Amplification and Strand Immobilization. Amplification wascarried out with exon 10 specific primers using standard PCR conditions(30 cycles: 1′@95° C., 1′@55° C., 2′@72° C.); the reverse primer was 5′labelled with biotin and column purified (Oligopurification Cartridge,Cruachem). After amplification the PCR products were purified by columnseparation (Qiagen Quickspin) and immobilized on streptavidin coatedmagnetic beads (DynaBeads, Dynal, Norway) according to their standardprotocol; DNA was denatured using 0.1M NaOH and washed with 0.1M NaOH,1×B+W buffer and TE buffer to remove the non-biotinylated sense strand.

[0124] COSBE Conditions. The beads containing ligated antisense strandwere resuspended in 18 μl of Reaction mix (2 μl 10×Taq buffer, 1 μL (1unit) Taq Polymerase, 2 μL of 2 mM dGTP, and 13 μL H₂O) and incubated at80° C. for 5′ before the addition of Reaction mix 2 (100 ng each ofCOSBE primers). The temperature was reduced to 60° C. and the mixturesincubated for 5′ annealing/extension period; the beads were then washedin 25 mM triethylammonium acetate (TEAA) followed by 50 mM ammoniumcitrate.

[0125] Primer Sequences. All primers were synthesized on a PerseptiveBiosystems Expedite 8900 DNA Synthesizer using conventionalphosphoramidite chemistry (Sinha et al. (1984) Nucleic Acids Res. 12:4539. COSBE primers (both containing an intentional mismatch one basebefore the 3′-terminus) were those used in a previous ARMS study (Ferrieet al., (1992) Am J Hum Genet 51: 251-262) with the exception that twobases were removed from the 5′-end of the normal:

[0126] Ex 10 PCR (Forward): 5′-BIO-GCA AGT GAA TCC TGA GCG TG-3′(SEQ.ID.No. 1)

[0127] Ex 10 PCR (Reverse): 5′-GTG TGA AGG GTT CAT ATG C-3′ (SEQ.ID.No.2)

[0128] COSBE ΔF508-N 5′-ATC TAT ATT CAT CAT AGG AAA CAC CAC A-3′(28-mer) (SEQ.ID.No. 3)

[0129] COSBE ΔF508-M 5′-GTA TCT ATA TTC ATC ATA GGA AAC ACC ATT-3′(30-mer) (SEQ.ID.No. 4).

[0130] Mass Spectrometry. After washing, beads were resuspended in 1 μl18 Mohm/cm H₂O. 300 nL each of matrix (Wu et al. (1993) Rapid CommunMass Spectrom 7: 142-146) solution (0.7 M 3-hydroxypicolinic acid, 0.7 Mdibasic ammonium citrate in 1:1 H₂O:CH₃CN) and resuspended beads (Tanget al. (1995) Rapid Commun Mass Spectrom 8: 727-730) were mixed on asample target and allowed to air dry. Up to 20 samples were spotted on aprobe target disk for introduction into the source region of anunmodified Thermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOFoperated in reflectron mode with 5 and 20 kV on the target andconversion dynode, respectively. Theoretical average molecular weights(M_(r)(calc)) were calculated from atomic compositions. Vendor-providedsoftware was used to determine peak centroids using externalcalibration; 1.08 Da has been subtracted from these to correct for thecharge-carrying proton mass to yield the test Mr(exp) values.

[0131] Scheme. Upon annealing to the bound template, the N and M primers(8508.6 and 9148.0 Da, respectively) are presented with dGTP; onlyprimers with proper Watson-Crick base paring at the variable (V)position are extended by the polymerase. Thus if V pairs with3′-terminal base of N, N is extended to a 8837.9 Da product (N+1).Likewise, if V is properly matched to the M terminus, M is extended to a9477.3 Da M+1 product.

Results

[0132] FIGS. 14-18 show the representative mass spectra of COSBEreaction products. Better results were obtained when PCR products werepurified before the biotinylated anti-sense strand was bound.

EXAMPLE 4 Differentiation of Human Apolipoprotein E Isoforms by MassSpectrometry

[0133] Apolipoprotein E (Apo E), a protein component of lipoproteins,plays an essential role in lipid metabolism. For example, it is involvedwith cholesterol transport, metabolism of lipoprotein particles,immunoregulation and activation of a number of lipolytic enzymes.

[0134] There are common isoforms of human Apo E (coded by E2, E3, and E4alleles). The most common is the E3 allele. The E2 allele has been shownto decrease the cholesterol level in plasma and therefore may have aprotective effect against the development of atherosclerosis. Finally,the E4 isoform has been correlated with increased levels of cholesterol,conferring predisposition to atherosclerosis. Therefore, the identity ofthe apo E allele of a particular individual is an important determinantof risk for the development of cardiovascular disease.

[0135] As shown in FIG. 19, a sample of DNA encoding apolipoprotein Ecan be obtained from a subject, amplified (e.g. via PCR); and the PCRproduct can be digested using an appropriate enzyme (e.g. Cfol). Therestriction digest obtained can then be analyzed by a variety of means.As shown in FIGS. 20A-20B, the three isotypes of apolipoprotein E (E2,E3 and E4 have different nucleic acid sequences and therefore also havedistinguishable molecular weight values.

[0136] As shown in FIGS. 21A-21C, different Apolipoprotein E genotypesexhibit different restriction patterns in a 3.5% MetPhor Agarose Gel or12% polyacrylamide gel. As shown in FIGS. 22A-22B and 23A-23B, thevarious apolipoprotein E genotypes can also be accurately and rapidlydetermined by mass spectrometry.

EXAMPLE 5 Detection of Hepatitis B Virus in Serum Samples Materials andMethods

[0137] Sample Preparation

[0138] Phenol/chloroform extraction of viral DNA and the final ethanolprecipitation was done according to standard protocols.

[0139] First PCR:

[0140] Each reaction was performed with 5 μl of the DNA preparation fromserum. 15 pmol of each primer and 2 units Taq DNA polymerase (PerkinElmer, Weiterstadt, Germany) were used. The final concentration of eachdNTP was 200 μM, and the final volume of the reaction was 50 μl. 10×PCRbuffer (Perkin Elmer, Weiterstadt, Germany) contained 100 mM Tris-HCl,pH 8.3, 500 mM KCl, 15 mM MgCl₂, and 0.01% gelatine (w/v). Primersequences: Primer 1: 5′-GCTTTGGGGCATGGACATTGACCCGTATAA-3′ (SEQ ID NO 5)Primer 2: 5′-CTGACTACTAATTCCCTGGATGCTGGGTCT-3′ (SEQ ID NO 6)

[0141] Nested PCR:

[0142] Each reaction was performed either with 1 μl of the firstreaction or with a 1:10 dilution of the first PCR as template,respectively. 100 pmol of each primer, 2.5 u Pfu(exo-) DNA polymerase(Stratagene, Heidelberg, Germany), a final concentration of 200 μM ofeach dNTPs and 5 μl 10×Pfu buffer (200 mM Tris-HCl, pH 8.75, 100 mM KCl,100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1% Triton X-100, and 1 mg/ml BSA,(Stratagene, Heidelberg, Germany) were used in a final volume of 50 μl.The reactions were performed in a thermocycler (OmniGene, MWG-Biotech,Ebersberg, Germany) using the following program: 92° C. for 1 minute,60° C. for 1 minute and 72° C. for 1 minute with 20 cycles. Thesequences of the HBV oligodeoxynucleotides (purchased HPLC-purified atMWG-Biotech, Ebersberg, Germany) are provided below.

[0143] HBV 13: 5′-TTGCCTGAGTGCAGTATGGT-3′ (SEQ.ID.No. 7)

[0144] HBV 15bio: Biotin-5′-AGCTCTATATCGGGAAGCCCT-3′ (SEQ.ID.No. 8)

[0145] Purification of PCR Products:

[0146] For the recording of each spectrum, one PCR, 50 μl, (performed asdescribed above) was used. Purification was done according to thefollowing procedure: Ultrafiltration was done using Ultrafree-MCfiltration units (Millipore, Eschborn, Germany) according to theprotocol of the provider with centrifugation at 8000 rpm for 20 minutes.25 μl (10 μg/μl) streptavidin Dynabeads (Dynal, Hamburg, Germany) wereprepared according to the instructions of the manufacturer andresuspended in 25 μl of B/W buffer (10 mM Tris-HC1, pH7.5, 1 mM EDTA, 2M NaC1). This suspension was added to the PCR samples still in thefiltration unit and the mixture was incubated with gentle shaking for 15minutes at ambient temperature. The suspension was transferred in a 1.5ml Eppendorf tube and the supernatant was removed with the aid of aMagnetic Particle Collector, MPC, (Dynal, Hamburg, Germany). The beadswere washed twice with 50 μl of 0.7 M ammonium citrate solution, pH 8.0(the supernatant was removed each time using the MPC). Cleavage from thebeads can be accomplished by using formamide at 90° C. The supernatantwas dried in a speedvac for about an hour and resuspended in 4 μl ofultrapure water (MilliQ UF plus Millipore, Eschborn, Germany). Thepreparation was used for MALDI-TOF MS analysis.

[0147] MALDI-TOR MS:

[0148] Half a microliter of the sample was pipetted onto the sampleholder, then immediately mixed with 0.5 μl matrix solution (0.7 M3-hydroxypicolinic acid, 50% acetonitrile, 70 mM ammonium citrate). Thismixture was dried at ambient temperature and introduced into the massspectrometer. All spectra were taken in positive ion mode using aFinnigan MAT Vision 2000 (Finnigan MAT, Bremen, Germany), equipped witha reflectron 5 keV ion source, 20 keV postacceleration) and a 337 nmnitrogen laser. Calibration was done with a mixture of a 40-mer and a100-mer. Each sample was measured with different laser energies. In thenegative samples, the PCR product was detected neither with less norwith higher laser energies. In the positive samples the PCR product wasdetected at different places of the sample spot and also with varyinglaser energies.

Results

[0149] A nested PCR system was used for the detection of HBV DNA inblood samples employing oligonucleotides complementary to the c regionof the HBV genome (primer 1: beginning at map position 1763, primer 2beginning at map position 2032 of the complementary strand) encoding theHBV core antigen (HBV cAg). DNA was isolated from patients serumaccording to standard protocols. A first PCR was performed with the DNAfrom these preparations using a first set of primers. If HBV DNA waspresent in the sample, a DNA fragment of 269 bp was generated.

[0150] In the second reaction, primers which were complementary to aregion within the PCR fragment generated in the first PCR were used. IfHBV-related PCR products were present in the first PCR, a DNA fragmentof 67 bp was generated (see FIG. 25A) in this nested PCR. The usage of anested PCR system for detection provides a high sensitivity and alsoserves as a specificity control for the external PCR (Rolfs, A. et al.,PCR: Clinical Diagnostics and Research, Springer, Heidelberg, 1992). Afurther advantage is that the amount of fragments generated in thesecond PCR is high enough to ensure an unproblematic detection althoughpurification losses cannot be avoided.

[0151] The samples were purified using ultrafiltration to remove theprimers prior to immobilization on streptavidin DynaBeads. Thispurification was done because the shorter primer fragments wereimmobilized in higher yield on the beads due to steric reasons. Theimmobilization was done directly on the ultrafiltration membrane toavoid substance losses due to unspecific absorption on the membrane.Following immobilization, the beads were washed with ammonium citrate toperform cation exchange (Pieles, U. et al., (1992) Nucleic Acids Res 21:3191-3196). The immobilized DNA was cleaved from the beads using 25%ammonia which allows cleavage of DNA from the beads in a very shorttime, but does not result in an introduction of sodium cations.

[0152] The nested PCRs and the MALDI TOF analysis were performed withoutknowing the results of serological analysis. Due to the unknown virustiter, each sample of the first PCR was used undiluted as template andin a 1:10 dilution, respectively.

[0153] Sample 1 was collected from a patient with chronic active HBVinfection, was positive in HBs and HBe-antigen tests, but negative in adot blot analysis. Sample 2 was a serum sample from a patient with anactive HBV infection and a massive viremia who was HBV positive in a dotblot analysis. Sample 3 was a denatured serum sample, therefore noserological analysis could be performed but an increased level oftransaminases indicating liver disease was detected. In autoradiographanalysis (FIG. 24), the first PCR of this sample was negative.Nevertheless, there was some evidence of HBV infection. This sample isof interest for MALDI-TOF analysis, because it demonstrates that evenlow-level amounts of PCR products can be detected after the purificationprocedure. Sample 4 was from a patient who was cured of HBV infection.Samples 5 and 6 were collected from patients with a chronic active HBVinfection.

[0154]FIG. 24 shows the results of a PAGE analysis of the nested PCRreaction. A PCR product is clearly revealed in samples 1, 2, 5 and 6. Insample 4 no PCR product was generated; it is indeed HBV negative,according to the serological analysis. Amplification artifacts arevisible in lanes 2, 5 and 6, if non-diluted template was used. Theseartifacts were not generated if the template was used in a 1:10dilution. In sample 3, PCR product was only detectable if the templatewas not diluted. The results of PAGE analysis are in agreement with thedata obtained by serological analysis except for sample 3 as discussedabove.

[0155]FIG. 25A shows a mass spectrum of a nested PCR product from samplenumber 1 generated and purified as described above. The signal at 20754Da represents the single stranded PCR product (calculated: 20735 Da, asthe average mass of both strands of the PCR product cleaved from thebeads). The mass difference of calculated and obtained mass is 19 Da(0.09%). As shown in FIG. 25A, sample number 1 generated a high amountof PCR product, resulting in an unambiguous detection.

[0156]FIG. 25B shows a spectrum obtained from sample number 3. Asdepicted in FIG. 24, the amount of PCR product generated in this sectionis significantly lower than that from sample number 1. Nevertheless, thePCR product is clearly revealed with a mass of 20751 Da (calculated20735). The mass difference is 16 Da (0.08%). The spectrum depicted inFIG. 25C was obtained from sample number 4 which is HBV negative (as isalso shown in FIG. 24). As expected no signals corresponding to the PCRproduct could be detected. All samples shown in FIGS. 25A-25C wereanalyzed with MALDI-TOF MS, whereby PCR product was detected in all HBVpositive samples, but not in the HBV negative samples. These resultswere reproduced in several independent experiments.

EXAMPLE 6 Analysis of Ligase Chain Reaction Products Via MALDI-TOF MassSpectrometry Materials and Methods

[0157] Oligodeoxynucleotides

[0158] Except for the biotinylated oligonucleotide, all otheroligonucleotides were synthesized in a 0.2 μmol scale on a MilliGen 7500DNA Synthesizer (Millipore, Bedford, Mass., USA) using theβ-cyanoethylphosphoamidite method (Sinha, N. D. et al., (1984) NucleicAcids Res. 12: 4539-4577). The oligodeoxynucleotides wereRP-HPLC-purified and deprotected according to standard protocols. Thebiotinylated oligodeoxynucleotide was purchased (HPLC-purified) fromBiometra, Gottingen, Germany).

[0159] Sequences and calculated masses of the oligonucleotides used:Oligodeoxynucleotide A: 5′-p-TTGTGCCACGCGGTTGGGAATGTA-3′ (SEQ.ID.No.9)(7521 Da) Oligodeoxynucleotide B: 5′-p-AGCAACGACTGTTTGCCCGCCAGTTG-3′(SEQ.ID.No.10) (7948 Da) Oligodeoxynucleotide C:5′-bio-TACATTCCCAACCGCGTGGCACAAC-3′ (SEQ.ID.No.11) (7960 Da)Oiigodeoxynucleotide D: 5′-p-AACTGGCGGGCAAACAGTCGTTGCT-3′ (SEQ.ID.No.12)(7708 Da)

[0160] 5′-Phosphorylation of Oligonucleotides A and D

[0161] This was performed with polynucleotide Kinase (Boehringer,Mannheim, Germany) according to published procedures; the5′-phosphorylated oligonucleotides were used unpurified for LCR.

[0162] Ligase Chain Reaction

[0163] The LCR was performed with Pfu DNA ligase and a ligase chainreaction kit (Stratagene, Heidelberg, Germany) containing two differentpBluescript KII phagemids, one carrying the wildtype form of the E.colilacI gene and the other one a mutant of the gene with a single pointmutation of bp 191 of the lacI gene.

[0164] The following LCR conditions were used for each reaction: 100 pgtemplate DNA (0.74 fmol) with 500 pg sonified salmon sperm DNA ascarrier, 25 ng (3.3 pmol) of each 5′-phosphorylated oligonucleotide, 20ng (2.5 pmol) of each non-phosphorylated oligonucleotide, and 4 u PfuDNA ligase in a final volume of 20 μl buffered by Pfu DNA ligasereaction buffer (Stratagene, Heidelberg, Germany). In a modelexperiment, a chemically synthesized ss 50-mer was used (1 fmol) astemplate, and in this case oligo C was also biotinylated. All reactionswere performed in a thermocycler (OmniGene, MWG-Biotech, Ebersberg,Germany) with the following program: 4 minutes 92° C., 2 minutes 60° C.and 25 cycles of 20 seconds 92° C., 40 seconds 60° C. Except for HPLCanalysis, the biotinylated ligation educt C was used. In a controlexperiment the biotinylated and non-biotinylated oligonucleotidesrevealed the same gel electrophoretic results. The reactions wereanalyzed on 7.5% polyacrylamide gels. Ligation product 1 (oligo A and B)calculated mass: 15450 Da; ligation product 2 (oligo C and D) calculatedmass: 15387 Da.

[0165] SMART-HPLC

[0166] Ion exchange HPLC (IE HPLC) was performed on the SMART-system(Pharmacia, Freibrug, Germany) using a Pharmacia Mono Q, PC 1.6/5column. Eluents were buffer A (25 mM Tris-HCl, 1 mM EDTA and 0.3 M NaClat pH 8.0) and buffer B (same as A, but 1 M NaCl). Starting with 100% Afor 5 minutes at a flow rate of 50 μl/min a gradient was applied from 0to 70% B in 30 minutes, then increased to 100% B in 2 minutes and heldat 100% B for 5 minutes. Two pooled LCR volumes (40 μl) performed witheither wildtype or mutant template were injected.

[0167] Sample Preparation for MALDI-TOF-MS

[0168] Preparation of immobilized DNA: For the recording of eachspectrum two LCRs (performed as described above) were pooled and diluted1:1 with 2×B/W buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl). Tothe samples 5 μl streptavidin DynaBeads (Dynal, Hamburg, Germany) wereadded and the mixture was allowed to bind with gentle shaking for 15minutes at ambient temperature. The supernatant was removed using aMagnetic Particle Collector, MPC (Dynal, Hamburg, Germany) and the beadswere washed twice with 50 μl of 0.7 M ammonium citrate solution (pH 8.0)(the supernatant was removed each time using the MPC). The beads wereresuspended in 1 μl of ultrapure water (MilliQ, Millipore, Bedford,Mass., USA). This suspension was directly used for MALDI-TOF-MS analysisas described below.

[0169] Combination of ultrafiltration and streptavidin DynaBeads: Forthe recording of spectrum two LCRs (performed as described above) werepooled, diluted 1:1 with 2×B/W buffer and concentrated with a 5000 NMWLUltrafree-MC filter unit (Millipore, Eschborn, Germany) according to theinstructions of the manufacturer. After concentration, the samples werewashed with 300 μl 1×B/W buffer and 5 μl streptavidin DynaBeads wereadded. The beads were washed once on the Ultrafree-MC filtration unitwith 300 μl of 1×B/W buffer and processed as described above. The beadswere resuspended in 30 to 50 μl of 1×B/W buffer and transferred in a 1.5ml Eppendorf tube. The supernatant was removed and the beads were washedtwice with 50 μl of 0.7 M ammonium citrate (pH 8.0). Finally, the beadswere washed once with 30 μl of acetone and resuspended in 1 μl ofultrapure water. The ligation mixture, after immobilization on thebeads, was used for MALDI-TOF-MS analysis as described below.

[0170] MALDI-TOF-MS

[0171] A suspension of streptavidin-coated magnetic beads with theimmobilized DNA was pipetted onto the sample holder, then immediatelymixed with 0.5 μl matrix solution (0.7 M 3-hydroxypicolinic acid in 50%acetonitrile, 70 mM ammonium citrate). This mixture was dried at ambienttemperature and introduced into the mass spectrometer. All spectra weretaken in positive ion mode using a Finnigan MAT Vision 2000 (FinniganMAT, Bremen, Germany), equipped with a reflectron (5 ke V ion source, 20keV postacceleration) and a nitrogen laser (337 nm). For the analysis ofPfu DNA ligase, 0.5 μl of the solution was mixed on the sample holderwith 1 μl of matrix solution and prepared as described above. For theanalysis of unpurified LCRs, 1 μl of an LCR was mixed with 1 μl matrixsolution.

Results and Discussion

[0172] The E. coli lacI gene served as a simple model system toinvestigate the suitability of MALDI-TOF-MS as a detection method forproducts generated in ligase chain reactions. This template systemcontains an E.coli lacI wildtype gene in a pBluescript KII phagemid andan E. coli lacI gene carrying a single point mutation at bp 191 (C to Ttransition) in the same phagemid. Four different oligonucleotides wereused, which were ligated only if the E. coli lacI wildtype gene waspresent (FIG. 26).

[0173] LCR conditions were optimized using Pfu DNA ligase to obtain atleast 1 pmol ligation product in each positive reaction. The ligationreactions were analyzed by polyacrylamide gel electrophoresis (PAGE) andHPLC on the SMART system (FIGS. 27, 28 and 29). FIG. 27 shows a PAGE ofa positive LCR with wildtype template (lane 1), a negative LCR withmutant template (1 and 2) and a negative control which contains enzyme,oligonucleotides and no template. The gel electrophoresis clearly showsthat the ligation product (50 bp) was produced only in the reaction withwildtype template whereas neither the template carrying the pointmutation nor the control reaction with salmon sperm DNA generatedamplification products. In FIG. 28, HPLC was used to analyze two pooledLCRs with wildtype template performed under the same conditions. Theligation product was clearly revealed. FIG. 29 shows the results of aHPLC in which two pooled negative LCRs with mutant template wereanalyzed. These chromatograms confirm the data shown in FIG. 27 and theresults taken together clearly demonstrate, that the system generatesligation products in a significant amount only if the wildtype templateis provided.

[0174] Appropriate control runs were performed to determine retentiontimes of the different compounds involved in the LCR experiments. Theseinclude the four oligonucleotides (A, B, C, and D), a synthetic ds50-mer (with the same sequence as the ligation product), the wildtypetemplate DNA, sonicated salmon sperm DNA and the Pfu DNA ligase inligation buffer.

[0175] In order to test which purification procedure should be usedbefore a LCR reaction can be analyzed by MALDI-TOF-MS, aliquots of anunpurified LCR (FIG. 30A) and aliquots of the enzyme stock solution(FIG. 30B) were analyzed with MALDI-TOF-MS. It turned out thatappropriate sample preparation is absolutely necessary since all signalsin the unpurified LCR correspond to signals obtained in the MALDI-TOF-MSanalysis of the Pfu DNA ligase. The calculated mass values of oligo Aand the ligation product are 7521 Da and 15450 Da, respectively. Thedata in FIGS. 30A-30B show that the enzyme solution leads to masssignals which do interfere with the expected signals of the ligationeducts and products and therefore makes an unambiguous signal assignmentimpossible. Furthermore, the spectra showed signals that the detergentTween 20 that is in the enzyme storage buffer influences thecrystallization behavior of the analyte/matrix mixture in an unfavorableway.

[0176] In one purification format streptavidin-coated magnetic beadswere used. As was shown in a recent paper, the direct desorption of DNAimmobilized by Watson-Crick base pairing to a complementary DNA fragmentcovalently bound to the beads is possible and the non-biotinylatedstrand will be desorbed exclusively (Tang, K. et al., Nucleic Acids Res.23: 3126-3131 (1995)). This approach in using immobilized ds DNA ensuresthat only the non-biotinylated strand will be desorbed. Ifnon-immobilized ds DNA is analyzed both strands are desorbed (Tang, K.et al., Rapid Comm. Mass Spectrom. 7: 183-186 (1994)) leading to broadsignals depending on the mass difference of the two strands. Therefore,employing this system for LCR, only the non-ligated oligonucleotide A,with a calculated mass of 7521 Da, and the ligation product from oligo Aand oligo B (calculated mass: 15450 Da) will be desorbed if oligo C isbiotinylated at the 5′ end and immobilized on steptavidin-coated beads.This results in a simple and unambiguous identification of the LCReducts and products.

[0177]FIG. 31A shows a MALDI-TOF mass spectrum obtained from two pooledLCRs (performed as described above) purified on streptavidin DynaBeadsand desorbed directly from the beads, indicating that the purificationmethod used was efficient (compared with FIGS. 30A-30B). A signal whichrepresents the unligated oligo A and a signal which corresponds to theligation product could be detected. The agreement between the calculatedand the experimentally found mass values is remarkable and allows anunambiguous peak assignment and accurate detection of the ligationproduct. In contrast, no ligation product but only oligo A could bedetected in the spectrum obtained from two pooled LCRs with mutatedtemplate (FIG. 31B). The specificity and selectivity of the LCRconditions and the sensitivity of the MALDI-TOF detection is furtherdemonstrated when performing the ligation reaction in the absence of aspecific template. FIG. 32 shows a spectrum obtained from two pooledLCRs in which only salmon sperm DNA was used as a negative control; onlyoligo A could be detected, as expected.

[0178] While the results shown in FIG. 31A can be correlated to lane 1of the gel in FIG. 27, the spectrum shown in FIG. 31B is equivalent tolane 2 in FIG. 27, and finally also the spectrum in FIG. 32 correspondsto lane 3 in FIG. 27. The results are in congruence with the HPLCanalysis presented in FIGS. 28 and 29. While both gel electrophoresis(FIG. 27) and HPLC (FIGS. 28 and 29) reveal either an excess or almostequal amounts of ligation product over ligation educts, the analysis byMALDI-TOF mass spectrometry produces a smaller signal for the ligationproduct (FIG. 31A).

[0179] The lower intensity of the ligation product signal could be dueto different desorption/ionization efficiencies between 24- and a50-mer. Since the T_(m) value of a duplex with 50 compared to 24 basepairs is significantly higher, more 24-mer could be desorbed. Areduction in signal intensity can also result from a higher degree offragmentation in the case of the longer oligonucleotides.

[0180] Regardless of the purification with streptavidin DynaBeads, FIG.32 reveals traces of Tween 20 in the region around 2000 Da. Substanceswith a viscous consistency negatively influence the process ofcrystallization and therefore can be detrimental to mass spectrometeranalysis. Tween 20 and also glycerol which are part of enzyme storagebuffers therefore should be removed entirely prior to mass spectrometeranalysis. For this reason an improved purification procedure whichincludes an additional ultrafiltration step prior to treatment withDynaBeads was investigated. Indeed, this sample purification resulted ina significant improvement of MALDI-TOF mass spectrometric performance.

[0181] FIGS. 33A-33B show spectra obtained from two pooled positive(33A) and negative (33B) LCRs, respectively. The positive reaction wasperformed with a chemically synthesized, single strand 50-mer astemplate with a sequence equivalent to the ligation product of oligo Cand D. Oligo C was 5′-biotinylated. Therefore the template was notdetected. As expected, only the ligation product of Oligo A and B(calculated mass 15450 Da) could be desorbed from the immobilized andligated oligo C and D. This newly generated DNA fragment is representedby the mass signal of 15448 Da in FIG. 33A. Compared to FIG. 31A, thisspectrum clearly shows that this method of sample preparation producessignals with improved resolution and intensity.

EXAMPLE 7 Mutation Detection by Solid Phase Oligo Base Extension of aPrimer and Analysis by MALDI-TOF Mass Spectrometry Summary

[0182] The solid-phase oligo base extension method detects pointmutations and small deletions as well as small insertions in amplifiedDNA. The method is based on the extension of a detection primer thatanneals adjacent to a variable nucleotide position on anaffinity-captured amplified template, using a DNA polymerase, a mixtureof three dNTPs, and the missing one dideoxy nucleotide. The resultingproducts are evaluated and resolved by MALDI-TOF mass spectrometrywithout further labeling procedures. The aim of the following experimentwas to determine mutant and wildtype alleles in a fast and reliablemanner.

Description of the Experiment

[0183] The method used a single detection primer followed by anoligo-nucleotide extension step to give products, differing in length bysome bases specific for mutant or wildtype alleles which can be easilyresolved by MALDI-TOF mass spectrometry. The method is described byusing as example the exon 10 of the CFTR-gene. Exon 10 of this geneleads, in the homozygous state, to the clinical phenotype of cysticfibrosis.

Materials and Methods

[0184] Genomic DNA

[0185] Genomic DNA were obtained from healthy individuals, individualshomozygous or heterozygous for the ΔF508 mutation, and one individualheterozygous for the 1506S mutation. The wildtype and mutant alleleswere confirmed by standard Sanger sequencing.

[0186] PCR Amplification of Exon 10 of the CFTR Gene

[0187] The primers for PCR amplification were CFEx10-F(5′-GCAAGTGAATCCTGAGCGTG-3′ (SEQ.ID.No. 13) located in intron 9 andbiotinylated) and CFEx10-R (5′-GTGTGAAGGGCGTG-3′, (SEQ.ID.No. 14)located in intron 10). Primers were used at a concentration of 8 pmol.Taq-polymerase including 10×buffer was purchased fromBoehringer-Mannheim and dTNPs were obtained from Pharmacia. The totalreaction volume was 50 μl. Cycling conditions for PCR were initially 5min. at 95° C., followed by 1 min. at 94° C., 45 sec at 53° C., and 30sec at 72° C. for 40 cycles with a final extension time of 5 min at 72°C.

[0188] Purification of the PCR Products

[0189] Amplification products were purified by using Qiagen's PCRpurification kit (No. 28106) according to manufacturer's instructions.The elution of the purified products from the column was done in 50 μlTE-buffer (10 mM Tris, 1 mM EDTA, pH 7.5).

[0190] Affinity-Capture and Denaturation of the Double Stranded DNA

[0191] 10 μl aliquots of the purified PCR product were transferred toone well of a streptavidin-coated microtiter plate (No. 1645684Boehringer-Mannheim or No. 95029262 Labsystems). Subsequently, 10 μlincubation buffer (80 mM sodium phosphate, 400 mM NaCl, 0.4% Tween 20,pH 7.5) and 30 μl water were added. After incubation for 1 hour at roomtemperature the wells were washed three times with 200 μl washing buffer(40 mM Tris, 1 mM EDTA, 50 mM NaCl, 0.1% Tween 20, pH 8.8). Todenaturate the double stranded DNA, the wells were treated with 100 μlof a 50 mM NaOH solution for 3 min. Hence, the wells were washed threetimes with 200 μl washing buffer.

[0192] Oligo Base Extension Reaction

[0193] The annealing of 25 pmol detection primer (CF508:5′-CTATATTCATCATAGGAAACACCA-3′ (SEQ ID No. 15)) was performed in 50 μlannealing buffer (20 mM Tris, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO, 1%Triton X-100, pH 8.75) at 50° C. for 10 min. The wells were washed threetimes with 200 μl washing buffer and once in 200 μl TE buffer. Theextension reaction was performed by using some components of the DNAsequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia.The total reaction volume was 45 μl, consisting of 21 μl sample, 6 μlSequenase-buffer, 3 μl 10 mM DTT solution, 4.5 μl of 0.5 mM of threedNTPs, 4.5 μl of 2 mM of the missing one ddNTP, 5.5 μl glycerol enzymedilution buffer, 0.25 μl Sequenase 2.0, and 0.25 μl pyrophosphatase. Thereaction was pipetted on ice and then incubated for 15 min. at roomtemperature and for 5 min. at 37° C. Hence, the wells were washed threetimes with 200 μl washing buffer and once with 60 μl of a 70 mMNH₄-citrate solution.

[0194] Denaturation and Precipitation of the Extended Primer

[0195] The extended primer was denatured in 50 μl 10% DMSO(dimethylsulfoxide) in water at 80° C. for 10 min. For precipitation, 10μl NH₄-acetate (pH 6.5), 0.5 μl glycogen (10 mg/ml water, Sigma No.G1765), and 100 μl absolute ethanol were added to the supernatant andincubated for 1 hour at room temperature. After centrifugation at 13,000g for 10 min., the pellet was washed in 70% ethanol and resuspended in 1μl 18 Mohm/cm H₂O water.

[0196] Sample Preparation and Analysis on MALDI-TOF Mass Spectrometry

[0197] Sample preparation was performed by mixing 0.3 μl each of matrixsolution (0.7 M 3-hydroxypicolinic acid, 0.07 M dibasic ammonium citratein 1:1 H₂O:CH₃CN) and of resuspended DNA/glycogen pellet on a sampletarget and allowed to air dry. Up to 20 samples were spotted on a probetarget disk for introduction into the source region of an unmodifiedThermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operatedin reflectron mode with 5 and 20 kV on the target and conversion dynode,respectively. Theoretical average molecular mass (M_(r)(calc)) werecalculated from atomic compositions; reported experimental Mr(M_(r)(exp)) values are those of the singly-protonated form, determinedusing external calibration.

Results

[0198] The aim of the experiment was to develop a fast and reliablemethod independent of exact stringencies for mutation detection thatleads to high quality and high throughput in the diagnosis of geneticdiseases. Therefore a special kind of DNA sequencing (oligo baseextension of one mutation detection primer) was combined with theevaluation of the resulting mini-sequencing products by matrix-assistedlaser desorption ionization (MALDI) mass spectrometry (MS). Thetime-of-flight (TOF) reflectron arrangement was chosen as a possiblemass measurement system. To prove this hypothesis, the examination wasperformed with exon 10 of the CFTR-gene, in which some mutations couldlead to the clinical phenotype of cystic fibrosis, the most commonmonogenetic disease in the Caucasian population.

[0199] The schematic presentations given in FIGS. 34A-34B show theexpected short sequencing products with the theoretically calculatedmolecular mass of the wildtype and various mutations of exon 10 of theCFTR-gene. The short sequencing products were produced using eitherddTTP (FIG. 34A) or ddCTP (FIG. 34B) to introduce a definitive sequencerelated stop in the nascent DNA strand. The MALDI-TOF-MS spectra ofhealthy, mutation heterozygous, and mutation homozygous individuals arepresented in FIGS. 35A-35B. All samples were confirmed by standardSanger sequencing which showed no discrepancy in comparison to the massspec analysis. The accuracy of the experimental measurements of thevarious molecular masses was within a range of minus 21.8 and plus 87.1dalton (Da) to the range expected. This is a definitive interpretationof the results allowed in each case. A further advantage of thisprocedure is the unambiguous detection of the ΔI507 mutation. In theddTTP reaction, the wildtype allele would be detected, whereas in theddCTP reaction the three base pair deletion would be disclosed.

[0200] The method described is highly suitable for the detection ofsingle point mutations or microlesions of DNA. Careful choice of themutation detection primers will open the window of multiplexing and leadto a high throughput including high quality in genetic diagnosis withoutany need for exact stringencies necessary in comparable allele-specificprocedures. Because of the uniqueness of the genetic information, theoligo base extension of mutation detection primer is applicable in eachdisease gene or polymorphic region in the genome like variable number oftandem repeats (VNTR) or other single nucleotide polymorphisms (e.g.,apolipoprotein E gene).

EXAMPLE 8 Detection of Polymerase Chain Reaction Products Containing7-Deazapurine Moieties With Matrix-Assisted Laser Desorption/IonizationTime-of-Flight (MALDI-TOF) Mass Spectrometry Materials and Methods

[0201] PCR Amplifications

[0202] The following oligodeoxynucleotide primers were eithersynthesized according to standard phosphoamidite chemistry (Sinha, N. D.et al., Tetrahdron Let. 24: 5843-5846 (1983); Sinha, N. D. et al.,Nucleic Acids Res. 12: 4539-4557 (1984)) on a Milligen 7500 DNAsynthesizer (Millipore, Bedford, Mass., USA) in 200 nmol scales orpurchased from MWG-Biotech (Ebersberg, Germany, primer 3) and Biometra(Goettingen, Germany, primers 6-7). primer 1: 5′-GTCACCCTCGACCTGCAG-3′;(SEQ.ID.No.16) primer 2: 5′-TTGTAAAACGACGGCCAGT-3′; (SEQ.ID.No.17)primer 3: 5′-CTTCCACCGCGATGTTGA-3′; (SEQ.ID.No.18) primer 4:5′-CAGGAAACAGCTATGAC-3′; (SEQ.ID.No.19) primer 5:5′-GTAAAACGACGGCCAGT-3′; (SEQ.ID.No.20) primer 6:5′-GTCACCCTCGACCTGCAgC-3′ (g: RiboG); (SEQ.ID.No.21) primer 7:5′-GTTGTAAAACGAGGGCCAgT-3′ (g: RiboG). (SEQ.ID.No.22)

[0203] The 99-mer and 200-mer DNA strands (modified and unmodified) aswell as the ribo- and 7-deaza-modified 100-mer were amplified from pRFc1DNA (10 ng, generously supplied S. Feyerabend, University of Hamburg) in100 μl reaction volume containing 10 mmol/L KCl, 10 mmol/L (NH₄)₂SO₄, 20mmol/L Tris HCl (pH=8.8), 2 mmol/L MgSO₄, (exo(−)Pseudococcus furiosus(Pfu)-Buffer, Pharmacia, Freiburg, Germany), 0.2 mmol/L each dNTP(Pharmacia, Freiburg, Germany), 1 μl of each primer and 1 unit ofexo(−)Pfu DNA polymerase (Stratagene, Heidelberg, Germany).

[0204] For the 99-mer primers 1 and 2, for the 200-mer primers 1 and 3and for the 100-mer primers 6 and 7 were used. To obtain 7-deazapurinemodified nucleic acids, during PCR-amplification dATP and dGTP werereplaced with 7-deaza-dATP and 7-deaza-dGTP. The reaction was performedin a thermal cycler (OmniGene, MWG-Biotech, Ebersberg, Germany) usingthe cycle: denaturation at 95° C. for 1 min., annealing at 51° C. for 1min. and extension at 72° C. for 1 min. For all PCRs the number ofreaction cycles was 30. The reaction was allowed to extend foradditional 10 min. at 72° C. after the last cycle.

[0205] The 103-mer DNA strands (modified and unmodified) were amplifiedfrom M13mp18 RFI DNA (100 ng, Pharmacia, Freiburg, Germany) in 100 μlreaction volume using primers 4 and 5; all other concentrations wereunchanged. The reaction was performed using the cycle: denaturation at95° C. for 1 min., annealing at 40° C. for 1 min., and extension at 72°C. for 1 min. After 30 cycles for the unmodified and 40 cycles for themodified 103-mer respectively, the samples were incubated for anadditional 10 min. at 72° C.

[0206] Synthesis of 5′-[³²P]-Labeled PCR-Primers

[0207] Primers 1 and 4 were 5′-[³²P]-labeled employing T4-polynucleotidekinase (Epicentre Technologies) and(γ-³²P)-ATP. (BLU/NGG/502A, Dupont,Germany) according to the protocols of the manufacturer. The reactionswere performed substituting 10% of primer 1 and 4 in PCR with thelabeled primers under otherwise unchanged reaction conditions. Theamplified DNAs were separated by gel electrophoresis on a 10%polyacrylamide gel. The appropriate bands were excised and counted on aPackard TRI-CARB 460C liquid scintillation system (Packard, Conn., USA).

[0208] Primer-Cleavage From Ribo-Modified PCR-Product

[0209] The amplified DNA was purified using Ultrafree-MC filter units(30,000 NMWL), it was then redissolved in 100 μl of 0.2 mol/L NaOH andheated at 95° C. for 25 minutes. The solution was then acidified withHCl (1 mol/L) and further purified for MALDI-TOF analysis employingUltrafree-MC filter units (30,000 NMWL) as described below.

[0210] Purification of PCR Products

[0211] All samples were purified and concentrated using Ultrafree-MCunits 30,000 NMWL (Millipore, Eschborn, Germany) according to themanufacturer's description. After lyophilization, PCR products wereredissolved in 5 μl (3 μl for the 200-mer) of ultrapure water. Thisanalyte solution was directly used for MALDI-TOF measurements.

[0212] MALDI-TOF MS

[0213] Aliquots of 0.5 μl of analyte solution and 0.5 μl of matrixsolution (0.7 mol/L 3-HPA and 0.07 mol/L ammonium citrate inacetonitrile/water (1:1, v/v) were mixed on a flat metallic samplesupport. After drying at ambient temperature the sample was introducedinto the mass spectrometer for analysis. The MALDI-TOF mass spectrometerused was a Finnigan MAT Vision 2000 (Finnigan MAT, Bremen, Germany).Spectra were recorded in the positive ion reflectron mode with a 5 keVion source and 20 keV postacceleration. The instrument was equipped witha nitrogen laser (337 nm wavelength). The vacuum of the system was3-4•10⁻⁸ hPa in the analyzer region and 1-4•10⁻⁷ hPa in the sourceregion. Spectra of modified and unmodified DNA samples were obtainedwith the same relative laser power; external calibration was performedwith a mixture of synthetic oligodeoxynucleotides (7- to 50-mer).

Results and Discussion

[0214] Enzymatic Synthesis of 7-Deazapurine-Nucleotide-ContainingNucleic Acids by PCR

[0215] In order to demonstrate the feasibility of MALDI-TOF MS for therapid, gel-free analysis of short PCR products and to investigate theeffect of 7-deazapurine modification of nucleic acids under MALDI-TOFconditions, two different primer-template systems were used tosynthesize DNA fragments. Sequences are displayed in FIGS. 36 and 37.While the two single strands of the 103-mer PCR product had nearly equalmasses (▾M=8 u), the two single strands of the 99-mer differed by 526 u.

[0216] Considering that 7-deaza purine nucleotide building blocks forchemical DNA synthesis are approximately 160 times more expensive thanregular ones (Product Information, Glen Research Corporation, Sterling,Va.) and their application in standard β-cyano-phosphoamidite chemistryis not trivial (Product Information, Glen Research Corporation,Sterling, Va.; Schneider, K. and B. T. Chait, Nucleic Acids Res. 23:1570 (1995)) the cost of 7-deaza-purine-modified primers would be veryhigh. Therefore, to increase the applicability and scope of the method,all PCRs were performed using unmodified oligonucleotide primers whichare routinely available. Substituting dATP and dGTP by c⁷-dATP andc⁷-dGTP in polymerase chain reaction led to products containingapproximately 80% 7-deaza-purine-modified nucleosides for the 99-mer and103-mer; and about 90% for the 200-mer, respectively. Table I shows thebase composition of all PCR products. TABLE I Base composition of the99-mer, 103-mer and 200-mer PCR amplification products (unmodified and7-deaza purine modified) c⁷- rel. DNA-fragments¹ C T A G deaza-Ac⁷-deaza-6 mod.² 200-mer s 54 34 56 56 — — — modified 200-mer s 54 34 65 50 51 90% 200-mer a 56 56 34 54 — — — modified 200-mer a 56 56 3 4 3150 92% 103-mer s 28 23 24 28 — — — modified 103-mer s 28 23 6 5 18 2379% 103-mer a 28 24 23 28 — — — modified 103-mer a 28 24 7 4 16 24 78%99-mer s 34 21 24 20 — — — modified 99-mer s 34 21 6 5 18 15 75% 99-mera 20 24 21 34 — — — modified 99-mer a 20 24 3 4 18 30 87%

[0217] However, it remained to be determined whether 80-90%7-deazapurine modification is sufficient for accurate mass spectrometerdetection. It was therefore important to determine whether all purinenucleotides could be substituted during the enzymatic amplificationstep. This was not trivial since it had been shown that c⁷-dATP cannotfully replace dATP in PCR if Taq DNA polymerase is employed (Seela, F.and A. Roelling, Nucleic Acids Res. 20: 55-61 (1992)). Fortunately wefound that exo(−)Pfu DNA polymerase indeed could accept c⁷-dATP andc⁷-dGTP in the absence of unmodified purine triphosphates. However, theincorporation was less efficient leading to a lower yield of PCR product(FIG. 38). Ethidium-bromide stains by intercalation with the stackedbases of the DNA-doublestrand. Therefore lower band intensities in theethidium-bromide stained gel might be artifacts since the modifiedDNA-strands do not necessarily need to give the same band intensities asthe unmodified ones.

[0218] To verify these results, the PCRs with [³²P]-labeled primers wererepeated. The autoradiogram (FIG. 39) clearly shows lower yields for themodified PCR-products. The bands were excised from the gel and counted.For all PCR products the yield of the modified nucleic acids was about50%, referring to the corresponding unmodified amplification product.Further experiments showed that exo(−)DeepVent and Vent DNA polymerasewere able to incorporate c⁷-dATP and c⁷-dGTP during PCR as well. Theoverall performance, however, turned out to be best for the exo(−)PfuDNA polymerase giving the least side products during amplification.Using all three polymerases, if was found that such PCRs employingc⁷-dATP and c⁷-dGTP instead of their isosteres showed fewerside-reactions giving a cleaner PCR-product. Decreased occurrence ofamplification side products may be explained by a reduction of primermismatches due to a lower stability of the complex formed from theprimer and the 7-deaza-purine-containing template which is synthesizedduring PCR. Decreased melting point for DNA duplexes containing7-deaza-purine have been described (Mizusawa, S. et al., Nucleic AcidsRes. 14: 1319-1324 (1986)). In addition to the three polymerasesspecified above (exo(−) Deep Vent DNA polymerase, Vent DNA polymeraseand exo(−) (Pfu) DNA polymerase), it is anticipated that otherpolymerases, such as the Large Klenow fragment of E.coli DNA polymerase,Sequenase, Taq DNA polymerase and U AmpliTaq DNA polymerase can be used.In addition, where RNA is the template, RNA polymerases, such as the SP6or the T7 RNA polymerase, must be used.

[0219] MALDI-TOF Mass Spectrometry of Modified and Unmodified PCRProducts

[0220] The 99-mer, 103-mer and 200-mer PCR products were analyzed byMALDI-TOF MS. Based on past experience, it was known that the degree ofdepurination depends on the laser energy used for desorption andionization of the analyte. Since the influence of 7-deazapurinemodification on fragmentation due to depurination was to beinvestigated, all spectra were measured at the same relative laserenergy.

[0221]FIGS. 40A and 40B show the mass spectra of the modified andunmodified 103-mer nucleic acids. In case of the modified 103-mer,fragmentation causes a broad (M+H)⁺ signal. The maximum of the peak isshifted to lower masses so that the assigned mass represents a meanvalue of the (M+H)⁺ signal and signals of fragmented ions, rather thanthe (M+H)⁺ signal itself. Although the modified 103-mer still containsabout 20% A and G from the oligonucleotide primers, it shows lessfragmentation which is featured by much more narrow and symmetricsignals. Peak tailing, especially on the lower mass side due todepurination, is substantially reduced. Hence, the difference betweenmeasured and calculated mass is strongly reduced although it is stillbelow the expected mass. For the unmodified sample, a (M+H)⁺ signal of31670 was observed, which is a 97 u or 0.3% difference to the calculatedmass, while in the case of the modified sample, this mass differencediminished to 10 u or 0.03% (31713 u found, 31723 u calculated). Theseobservations are verified by a significant increase in mass resolutionof the (M+H)⁺ signal of the two signal strands (m/Δm=67 as opposed to 18for the unmodified sample with Δm=full width at half maximum, fwhm).Because of the low mass difference between the two single strands (8u),their individual signals were not resolved.

[0222] With the results of the 99 base pair DNA fragments, the effectsof increased mass resolution for 7-deazapurine-containing DNA becomeseven more evident. The two single strands in the unmodified sample werenot resolved even though the mass difference between the two strands ofthe PCR product was very high with 526 u due to unequal distribution ofpurines and pyrimidines (FIGS. 41A1 and 41A2). In contrast to this, themodified DNA showed distinct peaks for the two single strands (FIGS.41B1 and 41B2) which makes the superiority of this approach for thedetermination of molecular weights to gel electrophoretic methods evenmore profound. Although base line resolution was not obtained, theindividual masses were able to be assigned with an accuracy of 0.1%(Δm=27 u for the lighter (calc. mass=30224 u) and Δm=14 u for theheavier strand (calc. mass=30750 u)). Again, it was found that the fullwidth at half maximum was substantially decreased for the 7-deazapurinecontaining sample.

[0223] In case of the 99-mer and the 103-mer, the 7-deazapurinecontaining nucleic acids seem to give higher sensitivity despite thefact that they still contain about 20% unmodified purine nucleotides. Toget comparable signal-to-noise ratio at similar intensities for the(M+H)⁺ signals, the unmodified 99-mer required 20 laser shots incontrast to 12 for the modified one and the 103-mer required 12 shotsfor the unmodified sample as opposed to three for the 7-deazapurinenucleoside-containing PCR product.

[0224] Comparing the spectra of the modified and unmodified 200-meramplicons, improved mass resolution was again found for the7-deazapurine-containing sample as well as increased signal intensities(FIGS. 42A and 42B). While the signal of the single strands predominatesin the spectrum of the modified sample the DNA-duplex and dimers of thesingle strands gave the strongest signal for the unmodified sample.

[0225] A complete 7-deaza purine modification of nucleic acids may beachieved either using modified primers in PCR or cleaving the unmodifiedprimers from the partially modified PCR product. Since disadvantages areassociated with modified primers, as described above, a 100-mer wassynthesized using primers with a ribo-modification. The primers werecleaved hydrolytically with NaOH according to a method developed earlierin our laboratory (Köster et al., Z. Physiol. Chem. 359: 1579-1589(1978)). Both hydrolyzed PCR product as well as the two released primerscould be detected, together with a small signal from residual uncleaved100-mer. This procedure is especially useful for the MALDI-TOF analysisof very short PCR-products since the share of unmodified purinesoriginating from the primer increases with decreasing length of theamplified sequence.

[0226] The remarkable properties of 7-deazapurine modified nucleic acidscan be explained by either more effective desorption and/or ionization,increased ion stability and/or a lower denaturation energy of the doublestranded purine-modified nucleic acid. The exchange of the N-7 for amethine group results in the loss of one acceptor for a hydrogen bondwhich influences the ability of the nucleic acid to form secondarystructures due to non-Watson-Crick base pairing (Seela, F. and A. Kehne,Biochemistry 26: 2232-2238 (1987)), which should be a reason for betterdesorption during the MALDI process. In addition to this, the aromaticsystem of 7-deazapurine has a lower electron density that weakensWatson-Crick base pairing resulting in a decreased melting point(Mizusawa, S. et al., Nucleic Acids Res. 14: 1319-1324 (1986)) of thedouble-strand. This effect may decrease the energy needed fordenaturation of the duplex in the MALDI process. These aspects as wellas the loss of a site which probably will carry a positive charge on theN-7 nitrogen renders the 7-deazapurine-modified nucleic acid less polarand may promote the effectiveness of desorption.

[0227] Because of the absence of N-7 as proton acceptor and thedecreased polarization of the C-N bond in 7-deazapurine nucleosides,depurination following the mechanisms established for hydrolysis insolution is prevented. Although a direct correlation of reactions insolution and in the gas phase is problematic, less fragmentation due todepurination of the modified nucleic acids can be expected in the MALDIprocess. Depurination may either be accompanied by loss of charge, whichdecreases the total yield of charged species, or it may produce chargedfragmentation products, which decreases the intensity of thenon-fragmented molecular ion signal.

[0228] The observation of both increased sensitivity and decreased peaktailing of the (M+H)⁺ signals on the lower mass side due to decreasedfragmentation of the 7-deazapurine-containing samples indicate that theN-7 atom indeed is essential for the mechanism of depurination in theMALDI-TOF process. In conclusion, 7-deazapurine-containing nucleic acidsshow distinctly increased ion-stability and sensitivity under MALDI-TOFconditions and therefore provide for higher mass accuracy and massresolution.

EXAMPLE 9 Solid State Sequencing and Mass Spectrometer DetectionMaterials and Methods

[0229] Oligonucleotides were purchased from Operon Technologies(Alameda, Calif.) in an unpurified form. Sequencing reactions wereperformed on a solid surface using reagents from the sequencing kit forSequenase Version 2.0 (Amersham, Arlington Heights, Ill.). Sequencing a39-mer target Sequencing complex:5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A^(b))_(a)-3′ (SEQ.ID.No.23)(DNA 11683) 3′-TCAACACTGCATGT-5′ (PNA 16/DNA) (SEQ.ID.No.24)

[0230] In order to perform solid-state DNA sequencing, template strandDNA11683 was 3′-biotinylated by terminal deoxynucleotidyl transferase. A30 μl reaction, containing 60 pmol of DNA11683, 1.3 nmol of biotin14-dATP (GIBCO BRL, Grand Island, N.Y.), 30 units of terminaltransferase (Amersham, Arlington Heights, Ill.), and 1×reaction buffer(supplied with enzyme), was incubated at 37° C. for 1 hour. The reactionwas stopped by heat inactivation of the terminal transferase at 70° C.for 10 min. The resulting product was desalted by passing through aTE-10 spin column (Clontech). More than one molecule of biotin-14-dATPcould be added to the 3′-end of DNA11683. The biotinylated DNA11683 wasincubated with 0.3 mg of Dynal streptavidin beads in 30 μl 1×binding andwashing buffer at ambient temperature for 30 min. The beads were washedtwice with TE, resuspended in 30 μl TE, and a 10 μl aliquot (containing0.1 mg of beads) was used for sequencing reactions.

[0231] The 0.1 mg beads from previous step were resuspended in a 10 μlvolume containing 2 μl of 5×Sequenase buffer (200 mM Tris-HCl, pH 7.5,100 mM MgCl₂, and 250 mM NaCl) from the Sequenase kit and 5 pmol ofcorresponding primer PNA16/DNA. The annealing mixture was heated to 70°C. and allowed to cool slowly to room temperature over a 20-30 min. timeperiod. Then 1 μl 0.1 M dithiothreitol solution, 1 μl Mn buffer (0.15 Msodium isocitrate and 0.1 M MnCl₂), and 2 μl of diluted Sequenase (3.25units) were added. The reaction mixture was divided into four aliquotsof 3 μl each and mixed with termination mixes (each consists of 3 μl ofthe appropriate termination mix: 32 μM c7dATP, 32 μM dCTP, 32 μM c7dGTP,32 μM dTTP and 3.2 μM of one of the four ddTNPS, in 50 mM NaCl). Thereaction mixtures were incubated at 37° C. for 2 min. After thecompletion of extension, the beads were precipitated and the supernatantwas removed. The beads were washed twice and resuspended in TE and keptat 4° C.

[0232] Sequencing a 78-mer Target Sequencing complex:5′-AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGCTGC (SEQ.ID.No.25)TGGATGATCCGACGCATCAGATCTGG-(A^(b))_(n)-3′ (TNR.PLASM2)3′-CTACTAGGCTGCGTAGTC-5′(CM1) (SEQ.ID.NO.26)

[0233] The target TNR.PLASM2 was biotinylated and sequenced usingprocedures similar to those described in previous section (sequencing a39-mer target).

[0234] Sequencing a 15-mer Target With Partially-Duplex Probe Sequencingcomplex: (SEQ.ID.No.27) 5′-F-GATGATCCGACGCATCACAGCTC-3′ (SEQ.ID.No.28)3′-b-CTACTAGGCTGCGTAGTGTCGAGAACCTTGGCT-5′

[0235] CM1B3B was immobilized on DynaBeads M280 with streptavidin(Dynal, Norway) by incubating 60 pmol of CM1B3B with 0.3 magnetic beadsin 30 μl 1 M NaCl and TE (1×binding and washing buffer) at roomtemperature for 30 min. The beads were washed twice with TE, resuspendedin 30 μl TE, and a 10 or 20 μl aliquot (containing 0.1 or 0.2 mg ofbeads respectively) was used for sequencing reactions.

[0236] The duplex was formed by annealing a corresponding aliquot ofbeads from previous step with 10 pmol of DF11a5F (or 20 pmol of DF11a5Ffor 0.2 mg of beads) in a 9 μl volume containing 2 μl of 5×Sequenasebuffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl₂, and 250 mM NaCl) from theSequenase kit. The annealing mixture was heated to 65° C. and allowed tocool slowly to 37° C. over a 20-30 min time period. The duplex primerwas then mixed with 10 pmol of TS10 (20 pmol of TS10 for 0.2 mg ofbeads) in 1 μl volume, and the resulting mixture was further incubatedat 37° C. for 5 min., and room temperature for 5-10 min. Then 1 μl 0.1 Mdithiothreitol solution, 1 μl Mn buffer (0.15 M sodium isocitrate and0.1 M MnCl₂), and 2 μl of diluted Sequenase (3.25 units) were added. Thereaction mixture was divided into four aliquots of 3 μl each and mixedwith termination mixes (each consists of 4 μl of the appropriatetermination mix: 16 μM dATP, 16 μM dCTP, 16 μM dGTP, 16 μM dTTP and 1.6μM of one of the four ddNTPs, in 50 mM NaCl). The reaction mixtures wereincubated at room temperature for 5 min., and 37° C. for 5 min. Afterthe completion of extension, the beads were precipitated and thesupernatant was removed. The beads were resuspended in 20 μl TE and keptat 4° C. An aliquot of 2 μl (out of 20 μl) from each tube was taken andmixed with 8 μl of formamide, the resulting samples were denatured at90-95° C. for 5 min. and 2 μl (out of 10 μl total) was applied to an ALFDNA sequencer (Pharmacia, Piscataway, N.J.) using a 10% polyacrylamidegel containing 7 M urea and 0.6×TBE. The remaining aliquot was used forMALDI-TOF MS analysis.

[0237] MALDI Sample Preparation and Instrumentation

[0238] Before MALDI analysis, the sequencing-ladder-loaded magneticbeads were washed twice using 50 mM ammonium citrate and resuspended in0.5 μl pure water. The suspension was then loaded onto the sample targetof the mass spectrometer and 0.5 μl of saturated matrix solution(3-hydropicolinic acid (HPA):ammonium citrate=10:1 mole ratio in 50%acetonitrile) was added. The mixture was allowed to dry prior to massspectrometer analysis.

[0239] The reflectron TOFMS mass spectrometer (Vision 2000, FinniganMAT, Bremen, Germany) was used for analysis. 5 kV was applied in the ionsource and 20 kV was applied for postaccelaration. All spectra weretaken in the positive ion mode and a nitrogen laser was used. Normally,each spectrum was averaged for more than 100 shots and a standard25-point smoothing was applied.

Results and Discussion

[0240] Conventional Solid-State Sequencing

[0241] In conventional sequencing methods, a primer is directly annealedto the template and then extended and terminated in a Sanger dideoxysequencing. Normally, a biotinylated primer is used and the sequencingladders are captured by streptavidin-coated magnetic beads. Afterwashing, the products are eluted from the beads using EDTA andformamide. However, our previous findings indicated that only theannealed strand of a duplex is desorbed and the immobilized strandremains on the beads. Therefore, it is advantageous to immobilize thetemplate and anneal the primer. After the sequencing reaction andwashing, the beads with the immobilized template and annealed sequencingladder can be loaded directly onto the mass spectrometer target andmixed with matrix. In MALDI, only the annealed sequencing ladder will bedesorbed and ionized, and the immobilized template will remain on thetarget.

[0242] A 39-mer template (SEQ.ID.No. 23) was first biotinylated at the3′ end by adding biotin-14-dATP with terminal transferase. More than onebiotin-14-dATP molecule could be added by the enzyme. However, since thetemplate was immobilized and remained on the beads during MALDI, thenumber of biotin-14-dATP added would not affect the mass spectra. A14-mer primer (SEQ.ID.No. 29) was used for the solid-state sequencing.MALDI-TOF mass spectra of the four sequencing ladders are shown in FIGS.44A-44D and the expected theoretical values are shown in Table II. TABLEII 1 5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A^(B))_(n)-3′ 2                          3′-TCAACACTGCATGT-5 3                         3′-ATCAACACTGCATGT-5′ 4                        3′-CATCAACACTGCATGT-5′ 5                       3′-ACATCAACACTGCATGT-5′ 6                      3′-AACATCAACACTGCATGT-5′ 7                     3′-TAACATCAACACTGCATGT-5′ 8                    3′-ATAACATCAACACTGCATGT-5′ 9                   3′-GATAACATCAACACTGCATGT-5′ 10                  3′-GGATAACATCAACACTGCATGT-5′ 11                 3′-CGGATAACATCAACACTGCATGT-5′ 12                3′-CCGGATAACATCAACACTGCATGT-5′ 13               3′-CCCGGATAACATCAACACTGCATGT-5′ 14              3′-TCCCGGATAACATCAACACTGCATGT-5′ 15             3′-GTCCCGGATAACATCAACACTGCATGT-5′ 16            3′-CGTCCCGGATAACATCAACACTGCATGT-5′ 17           3′-ACGTCCCGGATAACATCAACACTGCATGT-5′ 18          3′-CACGTCCCGGATAACATCAACACTGCATGT-5′ 19         3′-CCACGTCCCGGATAACATCAACACTGCATGT-5′ 20        3′-ACCACGTCCCGGATAACATCAACACTGCATGT-5′ 21       3′-GACCACGTCCCGGATAACATCAACACTGCATGT-5′ 22      3′-GGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 23     3′-CGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 24    3′-CCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ u25   3′-ACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 26  3′-GACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ 27 3′-AGACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′ A-reaction C-reactionG-reaction T-reaction 1. 2. 4223.8 4223.8 4223.8 4223.8 3. 4521.1 4.4810.2 5. 5123.4 6. 5436.6 7. 5740.8 8. 6054.0 9. 6383.2 10. 6712.4 11.7001.6 12. 7290.8 13. 7580.0 14. 7884.2 15. 8213.4 16. 8502.6 17. 8815.818. 9105.0 19. 9394.2 20. 9707.4 21. 10036.6 22. 10365.8 23. 10655.0 24.10944.2 25. 11257.4 26. 11586.6 27. 11899.8

[0243] The sequencing reaction produced a relatively homogenous ladder,and the full-length sequence was determined easily. One peak around 5150appeared in all reactions was not identified. A possible explanation isthat a small portion of the template formed some kind of secondarystructure, such as a loop, which hindered Sequenase extension.Mis-in-corporation is of minor importance, since the intensity of thesepeaks were much lower than that of the sequencing ladders. Although7-deaza purines were used in the sequencing reaction, which couldstabilize the N-glycosidic bond and prevent depurination, minor baselosses were still observed since the primer was not substituted by7-deazapurines. The full length ladder, with a ddA at the 3′ end,appeared in the A reaction with an apparent mass of 11899.8. However, amore intense peak of 122 appeared in all four reactions and is likelydue to an addition of an extra nucleotide by the Sequenase enzyme.

[0244] The same technique could be used to sequence longer DNAfragments. A 78-mer template containing a CTG repeat (SEQ.ID.No. 25) was3′-biotinylated by adding biotin-14-dATP with terminal transferase. An18-mer primer (SEQ.ID.No. 26) was annealed right outside the CTG repeatso that the repeat could be sequenced immediately after primerextension. The four reactions were washed and analyzed by MALDI-TOF MSas usual. An example of the G-reaction is shown in FIG. 45 and theexpected sequencing ladder is shown in Table III with theoretical massvalues for each ladder component. All sequencing peaks were wellresolved except the last component (theoretical value 20577.4) wasindistinguishable from the background. Two neighboring sequencing peaks(a 62-mer and a 63-mer) were also separated indicating that suchsequencing analysis could be applicable to longer templates. Again, anaddition of an extra nucleotide by the Sequenase enzyme was observed inthis spectrum. This addition is not template specific and appeared inall four reactions which makes it easy to be identified. Compared to theprimer peak, the sequencing peaks were at much lower intensity in thelong template case. Further optimization of the sequencing reaction maybe required. TABLE IIIAAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG-(A^(B))_(n)-3′1                                                      3′-CTACTAGGCTGCGTAGTC-5′2                                                     3′-CCTACTAGGCTGCGTAGTC-5′3                                                    3′-ACCTACTAGGCTGCGTAGTC-5′4                                                   3′-GACCTACTAGGCTGCGTAGTC-5′5                                                  3′-CGACCTACTAGGCTGCGTAGTC-5′6                                                 3′-ACGACCTACTAGGCTGCGTAGTC-5′7                                                3′-GACGACCTACTAGGCTGCGTAGTC-5′8                                               3′-CGACGACCTACTAGGCTGCGTAGTC-5′9                                              3′-ACGACGACCTACTAGGCTGCGTAGTC-5′10                                             3′-GACGACGACCTACTAGGCTGCGTAGTC-5′11                                            3′-CGACGACGACCTACTAGGCTGCGTAGTC-5′12                                           3′-ACGACGACGACCTACTAGGCTGCGTAGTC-5′13                                          3′-GACGACGACGACCTACTAGGCTGCGTAGTC-5′14                                         3′-CGACGACGACGACCTACTAGGCTGCGTAGTC-5′15                                        3′-ACGACGACGACGACCTACTAGGCTGCGTAGTC-5′16                                       3′-GACGACGACGACGACCTACTAGGCTGCGTAGTC-5′17                                      3′-CGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′18                                     3′-ACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′19                                    3′-GACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′20                                   3′-CGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′21                                  3′-ACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′22                                 3′-GACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′23                                3′-CGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′24                               3′-ACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′25                              3′-GACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′26                             3′-TGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′27                            3′-CTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′28                           3′-ACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′29                          3′-CACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′30                         3′-GCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′31                        3′-CGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′32                       3′-TCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′33                      3′-ATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′34                     3′-AATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′35                    3′-CAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′36                    3′-CCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′37                   3′-GCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′38                  3′-AGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′39                 3′-AAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′40                3′-TAAGCCAATCGCACTGACGACGACGACGACGAFGACGACCTACTAGGCTGCGTAGTC-5′41               3′-CTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′42              3′-CCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′43             3′-CCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′44            3′-TCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′45           3′-GTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′46          3′-GGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′47         3′-TGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′48        3′-CTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′49       3′-ACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′50      3′-GACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′51     3′-AGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′52    3′-TAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTAGTAGGCTGCGTAGTC-5′53   3′-CTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′54  3′-TCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′55 3′-TTCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′ddATP ddCTP ddGTP ddTTP 1. 5491.6 5491.6 5491.6 5491.6 2. 5764.8 3.6078.0 4. 6407.2 5. 6696.4 6. 7009.6 7. 7338.8 8. 7628.0 9. 7941.2 10.8270.4 11. 8559.6 12. 8872.8 13. 9202.0 14. 9491.2 15. 9804.4 16.10133.6 17. 10422.88 18. 10736.0 19. 11065.2 20. 11354.4 21. 11667.6 22.11996.8 23. 12286.0 24. 12599.2 25. 12928.4 26. 13232.6 27. 13521.8 28.13835.0 29. 14124.2 30. 14453.4 31. 14742.6 32. 15046.8 33. 15360.0 34.15673.2 35. 15962.4 36. 16251.6 37. 16580.8 38. 16894.0 39. 17207.2 40.17511.4 41. 17800.6 42. 18189.8 43. 18379.0 44. 18683.2 45. 19012.4 46.19341.6 47. 19645.8 48. 19935.0 49. 20248.2 50. 20577.4 51. 20890.6 52.21194.4 53. 21484.0 54. 21788.2 55. 22092.4

[0245] Sequencing Using Duplex DNA Probes for Capturing and Priming

[0246] Duplex DNA probes with single-stranded overhang have beendemonstrated to be able to capture specific DNA templates and alsoserved as primers for sold-state sequencing. The scheme is shown in FIG.46. Stacking interactions between a duplex probe and a single-strandedtemplate allow only 5-base overhang to be to be sufficient forcapturing. Based on this format, a 5′ fluorescent-labeled 23-mer (5′-GATGAT CCG ACG CAT CAC AGC TC-3′) (SEQ.ID.No. 29)) was annealed to a3′-biotinylated 18-mer (5′-GTG ATG CCT CGG ATC ATC-3′) (SEQ.ID.No. 30)),leaving a 5-base overhang. A 15-mer template (5′-TCG GTT CCA AGA GCT-3′)(SEQ.ID.No. 31)) was captured by the duplex and sequencing reactionswere performed by extension of the 5-base overhang. MALDI-TOF massspectra of the reactions are shown in FIGS. 47A-47D. All sequencingpeaks were resolved, although at relatively low intensities. The lastpeak in each reaction is due to unspecific addition of one nucleotide tothe full length extension product by the Sequenase enzyme. Forcomparison, the same products were run on a conventional DNA sequencerand a stacking fluorogram of the results is shown in FIGS. 48A-48D. Ascan be seen from the Figures, the mass spectra had the same pattern asthe fluorogram with sequencing peaks at much lower intensity compared tothe 23-mer primer.

[0247] Improvements of MALDI-TOF Mass Spectrometry as a DectionTechnique

[0248] Sample distribution can be made more homogenous and signalintensity could potentially be increased by implementing the picolitervial technique. In practice, the samples can be loaded on small pitswith square openings of 100 μm size. The beads used in the solid statesequencing are less than 10 μm in diameter, so they should fit well inthe microliter vials. Microcrystals of matrix and DNA containing “sweetspots” will be confined in the vial. Since the laser spot size is about100 μm in diameter, it will cover the entire opening of the vial.Therefore, searching for sweet spots will be unnecessary and highrepetition-rate laser (e.g. ⇄10 Hz) can be used for acquiring spectra.An earlier report has shown that this device is capable of increasingthe detection sensitivity of peptides and proteins by several orders ofmagnitude compared to the conventional MALDI sample preparationtechnique.

[0249] Resolution of MALDI on DNA needs to be further improved in orderto extend the sequencing range beyond 100 bases. Currently, using3-HPA/ammonium citrate as matrix and a reflectron TOF mass spectrometerwith 5 kV ion source and 20 kV postacceleration, the resolution of therun-through peak in FIG. 33A (73-mer) is greater than 200 (FWHM) whichis enough for sequence determination in this case. This resolution isalso the highest reported for MALDI desorbed DNA ions above the 70-merrange. Use of the delayed extraction technique may further enhanceresolution.

[0250] All of the above-cited references, applications and publicationsare herein incorporated by reference.

Equivalents

[0251] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

1 33 20 base pairs nucleic acid single linear cDNA 1 GCAAGTGAATCCTGAGCGTG 20 19 base pairs nucleic acid single linear cDNA 2 GTGTGAAGGGTTCATATGC 19 28 base pairs nucleic acid single linear cDNA 3 ATCTATATTCATCATAGGAA ACACCACA 28 30 base pairs nucleic acid single linear cDNA 4GTATCTATAT TCATCATAGG AAACACCATT 30 30 base pairs nucleic acid singlelinear cDNA 5 GCTTTGGGGC ATGGACATTG ACCCGTATAA 30 30 base pairs nucleicacid single linear cDNA 6 CTGACTACTA ATTCCCTGGA TGCTGGGTCT 30 20 basepairs nucleic acid single linear cDNA 7 TTGCCTGAGT GCAGTATGGT 20 20 basepairs nucleic acid single linear cDNA 8 AGCTCTATAT CGGGAAGCCT 20 24 basepairs nucleic acid single linear cDNA 9 TTGTGCCACG CGGTTGGGAA TGTA 24 26base pairs nucleic acid single linear cDNA 10 AGCAACGACT GTTTGCCCGCCAGTTG 26 25 base pairs nucleic acid single linear cDNA 11 TACATTCCCAACCGCGTGGC ACAAC 25 25 base pairs nucleic acid single linear cDNA 12AACTGGCGGG CAAACAGTCG TTGCT 25 57 base pairs nucleic acid single linearcDNA 13 ACCATTAAAG AAAATATCAT CTTTGGTGTT TCCTATGATG AATATAGAAG CGTCATC57 24 base pairs nucleic acid single linear cDNA 14 ACCACAAAGGATACTACTTA TATC 24 29 base pairs nucleic acid single linear cDNA 15TAGAAACCAC AAAGGATACT ACTTATATC 29 26 base pairs nucleic acid singlelinear cDNA 16 TAACCACAAA GGATACTACT TATATC 26 29 base pairs nucleicacid single linear cDNA 17 TAGAAACCAC AAAGGATACT ACTTATATC 29 38 basepairs nucleic acid single linear cDNA 18 CTTTTATAGT AGAAACCACAAAGGATACTA CTTATATC 38 35 base pairs nucleic acid single linear cDNA 19CTTTTATAGT AACCACAAAG GATACTACTT ATATC 35 35 base pairs nucleic acidsingle linear cDNA 20 CTTTTATAGA AACCACAAAG GATACTACTT ATATC 35 31 basepairs nucleic acid single linear cDNA 21 CGTAGAAACC ACAAAGGATACTACTTATAT C 31 58 base pairs nucleic acid single linear cDNA 22GAATTACATT CCCAACCGCG TGGCACAACA ACTGGCGGGC AAACAGTCGT TGCTGATT 58 58base pairs nucleic acid single linear cDNA 23 AATCAGCAAC GACTGTTTGCCCGCCAGTTG TTGTGCCACG CGGTTGGGAA TGTAATTC 58 252 base pairs nucleic acidsingle linear cDNA 24 GGCACGGCTG TCCAAGGAGC TGCAGGCGGC GCAGGCCCGGCTGGGCGCGG ACATGGAGGA 60 CGTGTGCGCC GCCTGGTGCA GTACCGCGGC GAGGTGCAGGCCATGCTCGG CCAGAGCAC 120 GAGGAGCTGC GGGTGCGCCT CGCCTCCCAC CTGCGCAAGCTGCGTAAGCG GCTCCTCCG 180 GATGCCGATG ACCTGCAGAA GTCCCTGGCA GTGTACCAGGCCGGGGCCCG CGAGGGCGC 240 GAGCGCGGCC TC 252 110 base pairs nucleic acidsingle linear cDNA 25 GCAACATTTT GCTGCCGGTC ACGGTTCGAA CGTACGGACGTCCAGCTGAG ATCTCCTAGG 60 GGCCCATGGC TCGAGCTTAA GCATTAGTAC CAGTATCGACAAAGGACACA 110 110 base pairs nucleic acid single linear cDNA 26TGTGTCCTTT GTCGATACTG GTACTAATGC TTAAGCTCGA GCCATGGGCC CCTAGGAGAT 60CTCAGCTGGA CGTCCGTACG TTCGAACCGT GACCGGCAGC AAAATGTTGC 110 217 basepairs nucleic acid single linear cDNA 27 AACGTGCTGC CTTCCACCGCGATGTTGATG ATTATGTGTC TGAATTTGAT GGGGGCAGGC 60 GGCCCCCGTC TGTTTGTCGCGGGTCTGGTG TTGATGGTGG TTTCCTGCCT TGTCACCCT 120 GACCTGCAGC CCAAGCTTGGGATCCACCAC CATCACCATC ACTAATAATG CATGGGCTG 180 AGCCAATTGG CACTGGCCGTCGTTTTACAA CGTCGTG 217 217 base pairs nucleic acid single linear cDNA 28CACGACGTTG TAAAACGACG GCCAGTGCCA ATTGGCTGCA GCCCATGCAT TATTAGTGAT 60GGTGATGGTG GTGGATCCCA AGCTTGGGCT GCAGGTCGAG GGTGACAAGG CAGGAAACC 120CCATCAACAC CAGACCCGCG ACAAACAGAC GGGGGCCGCC TGCCCCCATC AAATTCAGA 180ACATAATCAT CAACATCGCG GTGGAAGGCA GCACGTT 217 17 base pairs nucleic acidsingle linear cDNA 29 GTAAAACGAC GGCCAGT 17 17 base pairs nucleic acidsingle linear cDNA 30 CAGGAAACAG CTATGAC 17 18 base pairs nucleic acidsingle linear cDNA 31 CTTCCACCGC GATGTTGA 18 19 base pairs nucleic acidsingle linear cDNA 32 TTGTAAAACG ACGGCCAGT 19 18 base pairs nucleic acidsingle linear cDNA 33 GTCACCCTCG ACCTGCAG 18

What is claimed is:
 1. A method for detecting mutations in a target nucleic acid, comprising: digesting a target nucleic acid molecule; capturing digested fragments on a solid support that comprises oligonucleotides complementary thereto; detecting hybrids and the molecular weights of captured fragments by mass spectrometry, thereby identifying mutations in the target nucleic acid molecule.
 2. The method of claim 1, wherein the target molecule is RNA.
 3. The method of claim 2, wherein the RNA is produced by transcription of a target DNA molecule.
 4. The method of claim 1, wherein the solid support comprises a flat surface.
 5. The method of claim 1, wherein the solid support comprises matrix for performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
 6. The method of claim 1, wherein the solid support is selected from the group consisting of glass fiber filters, glass surfaces and metal surfaces.
 7. The method of claim 1, wherein the solid support is selected from the group consisting of steel, gold, silver, aluminum, copper and silicon.
 8. The method of claim 1, wherein the solid support is silicon.
 9. The method of claim 1, wherein the solid support is a silicon wafer.
 10. The method of claim 1, wherein the oligonucleotides on the solid support are linked to the support via a linker or a bond cleavable under the conditions of mass spectrometric analysis,
 11. The method of claim 10, wherein the oligonucleotides are linked to the support via a linker or bond is photocleavable.
 12. The method of claim 1, wherein the oligonucleotide comprises oligoribonucleotides, oligodeoxyribonucleotides, nucleotide analogs, or protein nucleic acid (PNA).
 13. The method of claim 10, wherein the nucleotide analogs comprise a thio-modified phosphodiester or phosphotriester backbone.
 14. The method of claim 1, wherein the oligonucleotides are linked to the support by an irreversible bond.
 15. The method of claim 1, wherein the oligonucleotides are linked to the support by a disulfide bond, a biotin/streptavidin linkage, a heterobifunctional derivative of a trityl ether group, a leuvinyl group, an arginine-arginine bond, a lysine-lysine bond, a pyrophosphate bond and a charge transfer complex. 